MicroRNA Compositions and Methods for Enhancing Plant Resistance to Abiotic Stress

ABSTRACT

A method of increasing tolerance of a plant to an abiotic stress or increasing biomass, vigor or yield of a plant is disclosed. The method comprising upregulating within the plant an exogenous polynucleotide of a microRNA or a precursor thereof, wherein the microRNA is selected from the group consisting of miR-156, miR-169, miR-164, miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408, miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129, thereby increasing the tolerance of the plant to the abiotic stress or increasing the biomass, vigor or yield of the plant.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/514,056, filed on Jun. 6, 2012, which is a U.S. National StageApplication of International Application No. PCT/IB2010/055600, filed onDec. 6, 2010, which claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/267,052, filed on Dec. 6, 2009, which isincorporated by reference in its entirety herein.

INCORPORATION OF SEQUENCE LISTING

A sequence listing is contained in the file named “P34397US02_SEQ.txt”which is 782,167 bytes in size (measured in MS-Windows) and was createdon Dec. 7, 2016, comprises 498 nucleotide sequences, is providedherewith via the USPTO's EFS system and is herein incorporated byreference in its entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to microRNAsand, more particularly, but not exclusively, to the use of same oralteration of same for generation of plants with enhanced resistance toabiotic stress.

MicroRNAs (miRNAs) are small, endogenous RNAs that regulate geneexpression in plants and animals. In plants, they are processed fromstem-loop regions of long primary transcripts by a Dicer-like enzyme andare loaded into silencing complexes, where they generally directcleavage of complementary mRNAs. Although plant miRNAs have someconserved functions extending beyond development, the importance ofmiRNA-directed gene regulation during plant development is now becomingclear. MiRNAs are already known to play numerous crucial roles at eachmajor stage of development, typically at the core of gene regulatorynetworks, targeting genes that are themselves regulators. So far,microRNAs have been found to be involved in plant development,regulation of abiotic and biotic stress responses and hormone signaling(Jones-Rhoades et al., 2006, Ann Rev Plant Biol 57:19-53).

A commonly-used approach in identifying the function of novel genes isthrough loss-of-function mutant screening. In many cases, functionalredundancy exists between genes that are members of the same family.When this happens, a mutation in one gene member might have a reduced oreven non-existing phenotype and the mutant lines might not be identifiedin the screening.

Using microRNAs, multiple members of the same gene family can besilenced simultaneously, giving rise to much more intense phenotypes.This approach is also superior to RNA interference (RNAi) techniques, inwhich typically 100-800 bp fragments of the gene of interest form afold-back structure when expressed. These long fold-back RNAs form manydifferent small RNAs and prediction of small RNA targets other than theperfectly complementary intended targets is therefore very difficult.MicroRNAs, in contrast, are produced from precursors, which are normallyprocessed such that preferentially one single, stable small RNA isgenerated, thus significantly minimizing the “off-target” effect.

A second approach to functional screening is through over-expression ofgenes of interest and testing for their phenotypes. In many cases,attempting to over-express a gene which is under microRNA regulationresults in no significant increase in the gene transcript. This can beovercome either by expressing a microRNA-resistant version of the geneor by down-regulating the microRNA itself.

Abiotic stress refers to such conditions as water deficit or drought,heat, cold, high or low nutrient or salt levels, and high or low lightlevels. In particular, drought and salinity are serious problems inagriculture and result in annual yield losses of billions of dollarsworldwide. Many genes are involved in the responses to abiotic stress inplants, but there is only limited information on miRNAs involved inplant response and adaptation to abiotic stress.

With a growing world population, increasing demand for food, fuel andfiber, and a changing climate, agriculture faces unprecedentedchallenges. In any given year, large areas of cornfields in the UnitedStates may be affected by at least moderate drought. Farmers are seekingadvanced, biotechnology-based solutions to enable them to obtain stablehigh yields and give them the potential to reduce irrigation costs or togrow crops in areas where potable water is a limiting factor.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of increasing tolerance of a plant to anabiotic stress or increasing biomass, vigor or yield of a plant, themethod comprising upregulating within the plant an exogenouspolynucleotide of a microRNA or a precursor thereof, wherein themicroRNA is selected from the group consisting of miR-156, miR-169,miR-164, miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a,ath-miR408, miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129, therebyincreasing the tolerance of the plant to the abiotic stress orincreasing the biomass, vigor or yield of the plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of increasing tolerance of a plant to anabiotic stress or increasing biomass, vigor or yield of a plant, themethod comprising expressing within the plant an exogenouspolynucleotide encoding a nucleic acid agent capable of downregulatingexpression of a target gene of a microRNA or a precursor thereof,wherein the microRNA is selected from the group consisting of miR-156,miR-169, miR-164, miR-159, miR-167, miR-529, miR-168, ppt-miR395,sof-miR408a, ath-miR408, miR-1039, miR-1091, miR-1118, miR-1134 andmiR-1129, thereby increasing the tolerance of the plant to the abioticstress or increasing the biomass, vigor or yield of the plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of increasing tolerance of a plant to anabiotic stress or increasing biomass, vigor or yield of a plant, themethod comprising expressing within the plant an exogenouspolynucleotide encoding a nucleic acid agent capable of downregulatingexpression or activity of a microRNA or a precursor thereof, wherein themicroRNA is selected from the group consisting of miR-171, miR-172,miR-399, miR-854, miR-894, miR-160, miR-166, miR-390, ath-miR395a,smo-miR408, miR-397, miR-477, miR-528, miR-530, miR-535, miR-855,miR-894, miR-896, miR-901 and miR-1026, thereby increasing the toleranceof the plant to the abiotic stress or increasing the biomass, vigor oryield of the plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of increasing tolerance of a plant to anabiotic stress or increasing biomass, vigor or yield of a plant, themethod comprising expressing within the plant an exogenouspolynucleotide for upregulating expression of a target gene of amicroRNA or a precursor thereof, wherein the microRNA is selected fromthe group consisting of miR-171, miR-172, miR-399, miR-854, miR-894,miR-160, miR-166, miR-390, ath-miR395a, smo-miR408, miR-397, miR-477,miR-528, miR-530, miR-535, miR-855, miR-894, miR-896, miR-901 andmiR-1026, thereby increasing the tolerance of the plant to the abioticstress or increasing the biomass, vigor or yield of the plant.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct, comprising a polynucleotideat least 90% homologous to a nucleic acid sequence selected from thegroup consisting of miR-156, miR-169, miR-164, miR-159, miR-167,miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408, miR-1039,miR-1091, miR-1118, miR-1134 and miR-1129 or a precursor thereof,wherein the nucleic acid sequence is under a transcriptional control ofat least one promoter capable of directing transcription of thepolynucleotide in a host cell.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct, comprising a polynucleotideat least 90% homologous to a nucleic acid sequence selected from thegroup consisting of a target gene of miR-171, a target gene of miR-172,a target gene of miR-399, a target gene of miR-854, a target gene ofmiR-894, a target gene of miR-160, a target gene of miR-166, a targetgene of miR-390, a target gene of ath-miR395a, a target gene ofsmo-miR408, a target gene of miR-397, a target gene of miR-477, a targetgene of miR-528, a target gene of miR-530, a target gene of miR-535, atarget gene of miR-855, a target gene of miR-894, a target gene ofmiR-896, a target gene of miR-901 and a target gene of miR-1026, whereinthe nucleic acid sequence is under a transcriptional control of at leastone promoter capable of directing transcription of the polynucleotide ina host cell.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct, comprising a nucleic acidsequence for down-regulating an expression of a target gene of amicroRNA or a precursor thereof, wherein the target gene of the microRNAis selected from the group consisting of a target gene of miR-156, atarget gene of miR-169, a target gene of miR-164, a target gene ofmiR-159, a target gene of miR-167, a target gene of miR-529, a targetgene of miR-168, a target gene of ppt-miR395, a target gene ofsof-miR408a, a target gene of ath-miR408, a target gene of miR-1039, atarget gene of miR-1091, a target gene of miR-1118, a target gene ofmiR-1134 and a target gene of miR-1129, wherein the nucleic acidsequence is under a transcriptional control of at least one promotercapable of directing transcription of the polynucleotide in a host cell.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct, comprising a nucleic acidsequence for down-regulating an expression of a microRNA or a precursorthereof, wherein the microRNA is selected from the group consisting ofmiR-171, miR-172, miR-399, miR-854, miR-894, miR-160, miR-166, miR-390,ath-miR395a, smo-miR408, miR-397, miR-477, miR-528, miR-530, miR-535,miR-855, miR-894, miR-896, miR-901 and miR-1026, wherein the nucleicacid sequence is under a transcriptional control of at least onepromoter capable of directing transcription of the polynucleotide in ahost cell.

According to an aspect of some embodiments of the present inventionthere is provided a plant cell, or a plant or a portion thereof,comprising a nucleic acid nucleic acid construct comprising (i) apolynucleotide at least 90% homologous to a nucleic acid sequenceselected from the group consisting of miR-156, miR-169, miR-164,miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408,miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129 or a precursorthereof, where the nucleic acid sequence is under a transcriptionalcontrol of at least one promoter capable of directing transcription ofthe polynucleotide in a host cell; or (ii) a polynucleotide at least 90%homologous to a nucleic acid sequence selected from the group consistingof a target gene of miR-171, a target gene of miR-172, a target gene ofmiR-399, a target gene of miR-854, a target gene of miR-894, a targetgene of miR-160, a target gene of miR-166, a target gene of miR-390, atarget gene of ath-miR395a, a target gene of smo-miR408, a target geneof miR-397, a target gene of miR-477, a target gene of miR-528, a targetgene of miR-530, a target gene of miR-535, a target gene of miR-855, atarget gene of miR-894, a target gene of miR-896, a target gene ofmiR-901 and a target gene of miR-1026, where the nucleic acid sequenceis under a transcriptional control of at least one promoter capable ofdirecting transcription of the polynucleotide in a host cell; or (iii) anucleic acid sequence for down-regulating an expression of a target geneof a microRNA or a precursor thereof, where the target gene of themicroRNA is selected from the group consisting of a target gene ofmiR-156, a target gene of miR-169, a target gene of miR-164, a targetgene of miR-159, a target gene of miR-167, a target gene of miR-529, atarget gene of miR-168, a target gene of ppt-miR395, a target gene ofsof-miR408a, a target gene of ath-miR408, a target gene of miR-1039, atarget gene of miR-1091, a target gene of miR-1118, a target gene ofmiR-1134, and a target gene of miR-1129 where the nucleic acid sequenceis under a transcriptional control of at least one promoter capable ofdirecting transcription of the polynucleotide in a host cell; or (iv) anucleic acid sequence for down-regulating expression of a microRNA or aprecursor thereof, where the microRNA is selected from the groupconsisting of miR-171, miR-172, miR-399, miR-854, miR-894, miR-160,miR-166, miR-390, ath-miR395a, smo-miR408, miR-397, miR-477, miR-528,miR-530, miR-535, miR-855, miR-894, miR-896, miR-901 and miR-1026, wherethe nucleic acid sequence is under a transcriptional control of at leastone promoter capable of directing transcription of the polynucleotide ina host cell.

According to an aspect of some embodiments of the present inventionthere is provided a food or feed comprising a plant, or a portionthereof, comprising a nucleic acid construct that comprises (i) apolynucleotide at least 90% homologous to a nucleic acid sequenceselected from the group consisting of miR-156, miR-169, miR-164,miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408,miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129 or a precursorthereof, where the nucleic acid sequence is under a transcriptionalcontrol of at least one promoter capable of directing transcription ofthe polynucleotide in a host cell; or (ii) a polynucleotide at least 90%homologous to a nucleic acid sequence selected from the group consistingof a target gene of miR-171, a target gene of miR-172, a target gene ofmiR-399, a target gene of miR-854, a target gene of miR-894, a targetgene of miR-160, a target gene of miR-166, a target gene of miR-390, atarget gene of ath-miR395a, a target gene of smo-miR408, a target geneof miR-397, a target gene of miR-477, a target gene of miR-528, a targetgene of miR-530, a target gene of miR-535, a target gene of miR-855, atarget gene of miR-894, a target gene of miR-896, a target gene ofmiR-901 and a target gene of miR-1026, where the nucleic acid sequenceis under a transcriptional control of at least one promoter capable ofdirecting transcription of the polynucleotide in a host cell; or (iii) anucleic acid sequence for down-regulating an expression of a target geneof a microRNA or a precursor thereof, where the target gene of themicroRNA is selected from the group consisting of a target gene ofmiR-156, a target gene of miR-169, a target gene of miR-164, a targetgene of miR-159, a target gene of miR-167, a target gene of miR-529, atarget gene of miR-168, a target gene of ppt-miR395, a target gene ofsof-miR408a, a target gene of ath-miR408, a target gene of miR-1039, atarget gene of miR-1091, a target gene of miR-1118, a target gene ofmiR-1134, and a target gene of miR-1129 where the nucleic acid sequenceis under a transcriptional control of at least one promoter capable ofdirecting transcription of the polynucleotide in a host cell; or (iv) anucleic acid sequence for down-regulating expression of a microRNA or aprecursor thereof, where the microRNA is selected from the groupconsisting of miR-171, miR-172, miR-399, miR-854, miR-894, miR-160,miR-166, miR-390, ath-miR395a, smo-miR408, miR-397, miR-477, miR-528,miR-530, miR-535, miR-855, miR-894, miR-896, miR-901 and miR-1026, wherethe nucleic acid sequence is under a transcriptional control of at leastone promoter capable of directing transcription of the polynucleotide ina host cell.

According to an aspect of some embodiments of the present inventionthere is provided a method of evaluating a trait of a plant, the methodcomprising: (a) expressing in a plant or a portion thereof a nucleicacid construct comprising (i) a polynucleotide at least 90% homologousto a nucleic acid sequence selected from the group consisting ofmiR-156, miR-169, miR-164, miR-159, miR-167, miR-529, miR-168,ppt-miR395, sof-miR408a, ath-miR408, miR-1039, miR-1091, miR-1118,miR-1134 and miR-1129 or a precursor thereof, where the nucleic acidsequence is under a transcriptional control of at least one promotercapable of directing transcription of the polynucleotide in a host cell,or (ii) a polynucleotide at least 90% homologous to a nucleic acidsequence selected from the group consisting of a target gene of miR-171,a target gene of miR-172, a target gene of miR-399, a target gene ofmiR-854, a target gene of miR-894, a target gene of miR-160, a targetgene of miR-166, a target gene of miR-390, a target gene of ath-miR395a,a target gene of smo-miR408, a target gene of miR-397, a target gene ofmiR-477, a target gene of miR-528, a target gene of miR-530, a targetgene of miR-535, a target gene of miR-855, a target gene of miR-894, atarget gene of miR-896, a target gene of miR-901 and a target gene ofmiR-1026, where the nucleic acid sequence is under a transcriptionalcontrol of at least one promoter capable of directing transcription ofthe polynucleotide in a host cell, or (iii) a nucleic acid sequence fordown-regulating an expression of a target gene of a microRNA or aprecursor thereof, where the target gene of the microRNA is selectedfrom the group consisting of a target gene of miR-156, a target gene ofmiR-169, a target gene of miR-164, a target gene of miR-159, a targetgene of miR-167, a target gene of miR-529, a target gene of miR-168, atarget gene of ppt-miR395, a target gene of sof-miR408a, a target geneof ath-miR408, a target gene of miR-1039, a target gene of miR-1091, atarget gene of miR-1118, a target gene of miR-1134, and a target gene ofmiR-1129 where the nucleic acid sequence is under a transcriptionalcontrol of at least one promoter capable of directing transcription ofthe polynucleotide in a host cell, or (iv) a nucleic acid sequence fordown-regulating expression of a microRNA or a precursor thereof, wherethe microRNA is selected from the group consisting of miR-171, miR-172,miR-399, miR-854, miR-894, miR-160, miR-166, miR-390, ath-miR395a,smo-miR408, miR-397, miR-477, miR-528, miR-530, miR-535, miR-855,miR-894, miR-896, miR-901 and miR-1026, where the nucleic acid sequenceis under a transcriptional control of at least one promoter capable ofdirecting transcription of the polynucleotide in a host cell; and (b)evaluating a trait of a plant as compared to a wild type plant of thesame type; thereby evaluating the trait of the plant.

According to some embodiments of the invention, the upregulating iseffected by expressing within the plant the exogenous polynucleotide ofthe microRNA or the precursor thereof.

According to some embodiments of the invention, the method comprisesgrowing the plant under abiotic stress conditions.

According to some embodiments of the invention, the abiotic stress isselected from the group consisting of salinity, water deprivation, lowtemperature, high temperature, heavy metal toxicity, anaerobiosis,nutrient deficiency, nutrient excess, atmospheric pollution and UVirradiation.

According to some embodiments of the invention, the expressing iseffected by transforming a cell of the plant with the exogenouspolynucleotide.

According to some embodiments of the invention, the transforming iseffected by introducing into the cell of the plant a nucleic acidconstruct including the exogenous polynucleotide and at least onepromoter capable of directing transcription of the exogenouspolynucleotide in the cell of the plant.

According to some embodiments of the invention, the expressing iseffected by infecting the plant with a bacteria comprising the exogenouspolynucleotide.

According to some embodiments of the invention, the downregulatingactivity of the microRNA is effected by introducing into the plant atarget mimic or a micro-RNA resistant target which is not cleaved by themicroRNA.

According to some embodiments of the invention, the target mimic or themicro-RNA resistant target is essentially complementary to the microRNAprovided that one or more of following mismatches are allowed:

(a) a mismatch between the nucleotide at the 5′ end of the microRNA andthe corresponding nucleotide sequence in the target mimic or themicro-RNA resistant target;

(b) a mismatch between any one of the nucleotides in position 1 toposition 9 of the microRNA and the corresponding nucleotide sequence inthe target mimic or the micro-RNA resistant target; or

(c) three mismatches between any one of the nucleotides in position 12to position 21 of the microRNA and the corresponding nucleotide sequencein the target mimic or the micro-RNA resistant target provided thatthere are no more than two consecutive mismatches.

According to some embodiments of the invention, the target mimic or themicro-RNA resistant target is introduced into a cell of the plant in anucleic acid construct including a target gene and at least one promotercapable of directing transcription of the target polynucleotide in thecell of the plant.

According to some embodiments of the invention, the target gene of themicroRNA is as set forth in SEQ ID NOs: 195-341, 474-485.

According to some embodiments of the invention, the host cell comprisesa plant cell.

According to some embodiments of the invention, the target gene ofmiR-169 comprises a NF-YA8 protein.

According to some embodiments of the invention, the miR-156 is selectedfrom the group consisting of bna-miR156a, smo-miR156c, sbi-miR156d,smo-miR156d, vvi-miR156e, ath-miR156g, ptc-miR156k, zma-miR156k andosa-miR156l.

According to some embodiments of the invention, the miR-169 is selectedfrom the group consisting of ath-miR169a, osa-miR169a, sbi-miR169b,bna-miR169c, sbi-miR169c, ath-miR169d, osa-miR169e, bna-miR169g,sbi-miR169i, bna-miR169m, vvi-miR169m, ptc-miR169o, ptc-miR169q,ptc-miR169v and ptc-miR169x.

According to some embodiments of the invention, the miR-164 is selectedfrom the group consisting of osa-miR164a, sbi-miR164b, osa-miR164c,osa-miR164e and ptc-miR164f.

According to some embodiments of the invention, the miR-167 is selectedfrom the group consisting of ppt-miR167, bna-miR167a, ath-miR167c,ath-miR167d, ptc-miR167f and ptc-miR167h.

According to some embodiments of the invention, the miR-1039 comprisesppt-miR1039-3p.

According to some embodiments of the invention, the miR-168 is selectedfrom the group consisting of sbi-miR168 and gma-miR168.

According to some embodiments of the invention, the miR-159 is selectedfrom the group consisting of pta-miR159c, sof-miR159c, osa-miR159c andosa-miR159d.

According to some embodiments of the invention, the miR-529 is selectedfrom the group consisting of ppt-miR529a, ppt-miR529d, ppt-miR529e andppt-miR529g.

According to some embodiments of the invention, the miR-1118 comprisestae-miR1118.

According to some embodiments of the invention, the miR-1134 comprisestae-miR1134.

According to some embodiments of the invention, the miR-1129 comprisestae-miR1129.

According to some embodiments of the invention, the miR-1091 comprisessmo-miR1091.

According to some embodiments of the invention, the miR-171 is selectedfrom the group consisting of smo-miR171a, vvi-miR171a, ath-miR171b,sbi-miR171b, smo-miR171b, zma-miR171c, sbi-miR171e, sbi-miR171f,zma-miR171f and vvi-miR171i.

According to some embodiments of the invention, the miR-172 is selectedfrom the group consisting of gma-miR172a, ath-miR172c and zma-miR172e.

According to some embodiments of the invention, the miR-854 comprisesath-miR854a.

According to some embodiments of the invention, the miR-894 comprisesppt-miR894.

According to some embodiments of the invention, the miR-160 is selectedfrom the group consisting of ppt-miR160b and ppt-miR160c.

According to some embodiments of the invention, the miR-390 is selectedfrom the group consisting of osa-miR390 and ppt-miR390c.

According to some embodiments of the invention, the miR-399 is selectedfrom the group consisting of sbi-miR399a, sbi-miR399b and mtr-miR399d.

According to some embodiments of the invention, the miR-166 comprisessbi-miR166e. According to some embodiments of the invention, the miR-397is selected from the group consisting of bna-miR397a and ptc-miR397b.

According to some embodiments of the invention, the miR-477 comprisesppt-miR477a-3p.

According to some embodiments of the invention, the miR-528 comprisesosa-miR528.

According to some embodiments of the invention, the miR-530 comprisesosa-miR530-3p.

According to some embodiments of the invention, the miR-535 comprisesvvi-miR535a.

According to some embodiments of the invention, the miR-855 comprisesath-miR855.

According to some embodiments of the invention, the miR-896 comprisesppt-miR896.

According to some embodiments of the invention, the miR-901 comprisesppt-miR901.

According to some embodiments of the invention, the miR-1026 comprisesppt-miR1026a.

According to some embodiments of the invention, the portion comprises aplant seed.

According to some embodiments of the invention, the plant is adicotyledonous plant.

According to some embodiments of the invention, the plant is amonocotyledonous plant.

According to some embodiments of the invention, the plant comprisescorn.

According to some embodiments of the invention, the plant comprisessorghum.

According to some embodiments of the invention, the plant is selectedfrom the group consisting of Arabidopsis, sorghum, corn, tobacco,cauliflower, soybean, alfalfa, peach, white spruce, wheat, sugar beet,sunflower, sugarcane, cotton, barley, tomato, potato, oat, carrot andgrape.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. Some embodiments of the invention are hereindescribed, by way of example only, with reference to the accompanyingdrawings. With specific reference now to the drawings in detail, it isstressed that the particulars shown are by way of example and forpurposes of illustrative discussion of embodiments of the invention. Inthis regard, the description taken with the drawings makes apparent tothose skilled in the art how embodiments of the invention may bepracticed.

In the drawings:

FIG. 1 shows a target-mimic sequence which could potentially be targetedby a miRNA, but contains extra nucleotides, leading to the creation of abulge at a position sensitive to mismatches (see further explanation inExample 6 of the Examples section which follows).

FIGS. 2A-B are photographs depicting miR-156a (SEQ ID NO: 191) andmiR-169a (SEQ ID NO: 12) transgenic Arabidopsis thaliana plantscomprising enhanced drought tolerance compared to wild-type plants. FIG.2A depicts control (watered) trays; and FIG. 2B shows plants after 10days of de-hydration.

FIGS. 3A-B are photographs depicting miR-156a (SEQ ID NO: 191) andmiR-169a (SEQ ID NO: 12) transgenic Arabidopsis thaliana plantscomprising enhanced drought tolerance compared to wild-type plants. FIG.3A depicts control (watered) trays; and FIG. 3B shows plants after 10days of de-hydration.

FIG. 4 is a photograph depicting miR-156a (SEQ ID NO: 191), miR-169a(SEQ ID NO: 12) and wild-type Arabidopsis thaliana plants watered after10 days of de-hydration. The photograph was taken after 7 days ofregeneration. Of note, wild-type plants did not survive while transgenicplants survived and displayed characteristics similar to the wateredplants.

FIGS. 5A-C are photographs depicting miR-156a (SEQ ID NO: 191), miR-169a(SEQ ID NO: 12) and wild-type Arabidopsis thaliana plants watered after10 days of de-hydration. FIGS. 5A-B depict trays of mature wild-type andtransgenic plants after 10 days of drought. FIG. 5C depicts single potsfrom the same experiment. Of note, wild-type plants did not survivewhile transgenic plants survived and displayed characteristics similarto the watered plants.

FIGS. 6A-B are photographs depicting miR-169a (SEQ ID NO: 12) andwild-type Arabidopsis thaliana plants. FIG. 6A depicts the transgenicand wild-type plants after a 10 day drought; and FIG. 6B depicts thetransgenic and wild-type plants after 7 days of re-hydration (followingthe drought).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to microRNAsand, more particularly, but not exclusively, to the use of same oralteration of same for generation of plants with enhanced resistance toabiotic stress.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

While reducing some embodiments of the present invention to practice,the present inventor has uncovered novel microRNA molecules involved inenhanced tolerance of plants to abiotic stress. Moreover, the presentinventor has constructed nucleic acid vectors expressing these microRNAsand used same for generating transgenic plants with enhanced toleranceto abiotic stress.

Thus, as shown in the Examples section which follows, the presentinventor has grown corn and sorghum under abiotic stress conditions(drought or high salinity) and analyzed, by high throughput microarrayand PCR, the differential expression levels of microRNAs in theseplants. Specifically, the present inventor has unveiled specificmicroRNAs which are upregulated or downregulated in response to abioticstress conditions such as drought and salinity (see Tables 2-5, in theExamples section which follows) and revealed possible target peptides ofthese microRNAs (see Tables 6-7, in the Examples section whichfollows).). Moreover, the present inventor has generated transgenicArabidopsis thaliana plants expressing miR-156a or miR-169a which canwithstand severe drought and continue to grow and thrive similarly towatered plants the plants appear less dry then the control throughoutthe de-hydration, and after the re-hydration the plants are able torecover and continue to grow and flower, while the wild-type either doesnot survive or is unable to develop properly. (see FIGS. 2A-B, 3A-B, 4,5A-C and 6A-B). Accordingly, these microRNAs and their specific targetgenes may serve as powerful tools in the field of agriculture transgenictechnologies.

As used herein the term “tolerance” refers to the ability of a plant toendure an abiotic stress without exhibiting substantial physiological orphysical damage (e.g. alteration in metabolism, growth, viability and/orreproductivity of the plant).

The phrase “abiotic stress” as used herein refers to any adverse effecton metabolism, growth, viability and/or reproduction of a plant. Abioticstress can be induced by any of suboptimal environmental growthconditions such as, for example, water deficit or drought, flooding,freezing, low or high temperature, strong winds, heavy metal toxicity,anaerobiosis, high or low nutrient levels (e.g. nutrient deficiency),high or low salt levels (e.g. salinity), atmospheric pollution, high orlow light intensities (e.g. insufficient light) or UV irradiation.Abiotic stress may be a short term effect (e.g. acute effect, e.g.lasting for about a week) or alternatively may be persistent (e.g.chronic effect, e.g. lasting for example 10 days or more). The presentinvention contemplates situations in which there is a single abioticstress condition or alternatively situations in which two or moreabiotic stresses occur.

According to an exemplary embodiment the abiotic stress refers tosalinity.

According to another exemplary embodiment the abiotic stress refers todrought.

As used herein the phrase “biomass of a plant” or “plant biomass” refersto the amount (e.g., measured in grams of air-dry tissue) of a tissueproduced from the plant in a growing season. An increase in plantbiomass can be in the whole plant or in parts thereof such asaboveground (e.g. harvestable) parts, vegetative biomass, roots and/orseeds.

As used herein the phrase “vigor of a plant” or “plant vigor” refers tothe amount (e.g., measured by weight) of tissue produced by the plant ina given time. Increased vigor could determine or affect the plant yieldor the yield per growing time or growing area. In addition, early vigor(e.g. seed and/or seedling) results in improved field stand.

As used herein the phrase “yield of a plant” or “plant yield” refers tothe amount (e.g., as determined by weight or size) or quantity (e.g.,numbers) of tissues or organs produced per plant or per growing season.Increased yield of a plant can affect the economic benefit one canobtain from the plant in a certain growing area and/or growing time.

According to an exemplary embodiment the yield is measured by cellulosecontent.

According to another exemplary embodiment the yield is measured by oilcontent.

According to another exemplary embodiment the yield is measured byprotein content.

A plant yield can be affected by various parameters including, but notlimited to, plant biomass; plant vigor; plant growth rate; seed yield;seed or grain quantity; seed or grain quality; oil yield; content ofoil, starch and/or protein in harvested organs (e.g., seeds orvegetative parts of the plant); number of flowers (e.g. florets) perpanicle (e.g. expressed as a ratio of number of filled seeds over numberof primary panicles); harvest index; number of plants grown per area;number and size of harvested organs per plant and per area; number ofplants per growing area (e.g. density); number of harvested organs infield; total leaf area; carbon assimilation and carbon partitioning(e.g. the distribution/allocation of carbon within the plant);resistance to shade; number of harvestable organs (e.g. seeds), seedsper pod, weight per seed; and modified architecture [such as increasestalk diameter, thickness or improvement of physical properties (e.g.elasticity)].

A plant yield can be determined under stress (e.g., abiotic stress, asdescribed above) and/or non-stress (e.g. normal) conditions.

The phrase “non-stress conditions” refers to the growth conditions(e.g., water, temperature, light-dark cycles, humidity, saltconcentration, fertilizer concentration in soil, nutrient supply such asnitrogen, phosphorous and/or potassium), that do not significantly gobeyond the everyday climatic and other abiotic conditions that plantsmay encounter, and which allow optimal growth, metabolism, reproductionand/or viability of a plant at any stage in its life cycle (e.g., in acrop plant from seed to a mature plant and back to seed again). Personsskilled in the art are aware of normal soil conditions and climaticconditions for a given plant in a given geographic location. It shouldbe noted that while the non-stress conditions may include some mildvariations from the optimal conditions (which vary from one type/speciesof a plant to another), such variations do not cause the plant to ceasegrowing without the capacity to resume growth.

As used herein, the terms “seed” or “grain” refer to a small embryonicplant enclosed in a covering called the seed coat (e.g., usually withsome stored food), the product of the ripened ovule of gymnosperm andangiosperm plants which occurs after fertilization and some growthwithin the mother plant.

As used herein the term “increasing” refers to at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90% or greater increase in tolerance toabiotic stress, in yield, in biomass or in vigor of a plant, as comparedto a native or wild-type plants [i.e., plants not modified with thebiomolecules (polynucleotides or polypeptides) of the invention, e.g., anon-transformed plant of the same species which is grown under the samegrowth conditions as the transformed plant].

For example, tolerance to abiotic stress (e.g. tolerance to drought orsalinity) can be evaluated by determining the differences inphysiological and/or physical condition, including but not limited to,vigor, growth, size, or root length, or specifically, leaf color or leafarea size of the transgenic plant compared to a non-modified plant ofthe same species grown under the same conditions. Other techniques forevaluating tolerance to abiotic stress include, but are not limited to,measuring chlorophyll fluorescence, photosynthetic rates and gasexchange rates. Further assays for evaluating tolerance to abioticstress are provided hereinbelow and in the Examples section whichfollows.

Thus, according to one aspect of the present invention, there isprovided a method of increasing tolerance of a plant to an abioticstress or increasing biomass, vigor or yield of a plant, the methodcomprising upregulating within the plant an exogenous polynucleotide ofa microRNA or a precursor thereof, wherein the microRNA is selected fromthe group consisting of miR-156, miR-169, miR-164, miR-159, miR-167,miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408, miR-1039,miR-1091, miR-1118, miR-1134 and miR-1129, thereby increasing thetolerance of the plant to the abiotic stress or increasing the biomass,vigor or yield of the plant.

According to another aspect of the present invention, there is provideda method of increasing tolerance of a plant to an abiotic stress orincreasing biomass, vigor or yield of a plant, the method comprisingexpressing within the plant an exogenous polynucleotide encoding anucleic acid agent capable of downregulating expression of a target geneof a microRNA or a precursor thereof, wherein the microRNA is selectedfrom the group consisting of miR-156, miR-169, miR-164, miR-159,miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408,miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129, thereby increasingthe tolerance of the plant to the abiotic stress or increasing thebiomass, vigor or yield of the plant.

According to another aspect of the present invention, there is provideda method of increasing tolerance of a plant to an abiotic stress orincreasing biomass, vigor or yield of a plant, the method comprisingexpressing within the plant an exogenous polynucleotide encoding anucleic acid agent capable of downregulating expression or activity of amicroRNA or a precursor thereof, wherein the microRNA is selected fromthe group consisting of miR-171, miR-172, miR-399, miR-854, miR-894,miR-160, miR-166, miR-390, ath-miR395a, smo-miR408, miR-397, miR-477,miR-528, miR-530, miR-535, miR-855, miR-894, miR-896, miR-901 andmiR-1026, thereby increasing the tolerance of the plant to the abioticstress or increasing the biomass, vigor or yield of the plant.

According to another aspect of the present invention, there is provideda method of increasing tolerance of a plant to an abiotic stress orincreasing biomass, vigor or yield of a plant, the method comprisingexpressing within the plant an exogenous polynucleotide for upregulatingexpression of a target gene of a microRNA or a precursor thereof,wherein the microRNA is selected from the group consisting of miR-171,miR-172, miR-399, miR-854, miR-894, miR-160, miR-166, miR-390,ath-miR395a, smo-miR408, miR-397, miR-477, miR-528, miR-530, miR-535,miR-855, miR-894, miR-896, miR-901 and miR-1026, thereby increasing thetolerance of the plant to the abiotic stress or increasing the biomass,vigor or yield of the plant.

The method of the present invention is effected by expressing within aplant an exogenous polynucleotide encoding a microRNA or a precursorthereof, a target gene or a down-regulating agent of the target gene orof the miR, as explained below.

As used herein, the phrase “expressing within the plant an exogenouspolynucleotide” refers to upregulating the expression level of anexogenous polynucleotide within the plant e.g., by introducing theexogenous polynucleotide into a plant or plant cell and expressing byrecombinant means, as described in detail hereinbelow. According to aspecific embodiment, short nucleic acid sequences (e.g., miRNAs orprecursors thereof) can be introduced into the plant directly as nakedRNA and not under a plant promoter. This is especially advantageous whensynthetic modifications are introduced (for transient expression suchsequences are introduced using any method known in the art such as forexample, particle bombardment).

As used herein “expressing” refers to expression at the mRNA level(e.g., in the case of a miRNA or an agent which downregulates expressionas described below) or at the polypeptide level (e.g., in the case of atarget gene) of the desired exogenous polynucleotide.

As used herein, the phrase “exogenous polynucleotide” refers to aheterologous nucleic acid sequence which may not be naturally expressedwithin the plant or which overexpression in the plant is desired (i.e.,overexpression of an endogenous gene). The exogenous polynucleotide maybe introduced into the plant in a stable or transient manner, so as toproduce a ribonucleic acid (RNA) molecule and/or a polypeptide molecule.The exogenous polynucleotide may comprise a nucleic acid sequence whichis identical or partially homologous to an endogenous nucleic acidsequence expressed within the plant.

The term “endogenous” as used herein refers to any polynucleotide orpolypeptide which is present and/or naturally expressed within a plantor a cell thereof.

As used herein the term “polynucleotide” refers to a single or doublestranded nucleic acid sequence which is isolated and provided in theform of an RNA sequence, a complementary polynucleotide sequence (cDNA),a genomic polynucleotide sequence (e.g. sequence isolated from achromosome) and/or a composite polynucleotide sequences (e.g., acombination of the above). This term includes polynucleotides and/oroligonucleotides derived from naturally occurring nucleic acidsmolecules (e.g., RNA or DNA), synthetic polynucleotide and/oroligonucleotide molecules composed of naturally occurring bases, sugars,and covalent internucleoside linkages (e.g., backbone), as well assynthetic polynucleotides and/or oligonucleotides having non-naturallyoccurring portions, which function similarly to the respective naturallyoccurring portions.

The term “isolated” refers to at least partially separated from thenatural environment e.g., from a plant cell.

The polynucleotides of the present invention may be of varying lengths,for example, in case of a nucleic acid sequence encoding a target gene,the length of the polynucleotide may be in the range of about 500 to2000 nucleic acids. Alternatively, in case of a miRNA or a precursorthereof, the polynucleotide sequence may be of shorter length. Forexample, in case of a miRNA, the length of the polynucleotide of thepresent invention is optionally about 100 to 300 nucleotides, about 100nucleotides or less, about 90 nucleotides or less, about 80 nucleotidesor less, about 70 nucleotides or less, about 60 nucleotides or less,about 50 nucleotides or less, about 40 nucleotides or less, about 30nucleotides or less, e.g., 29 nucleotides, 28 nucleotides, 27nucleotides, 26 nucleotides, 25 nucleotides, 24 nucleotides, 23nucleotides, 22 nucleotides, 21 nucleotides, 20 nucleotides, 19nucleotides, 18 nucleotides, 17 nucleotides, 16 nucleotides, 15nucleotides, about between 12 and 24 nucleotides, about between 5-15,about, between 5-25, or about 20-22 nucleotides in length.

Nucleic acid sequences of the polypeptides of some embodiments of theinvention may be optimized for expression in a specific plant host.Examples of such sequence modifications include, but are not limited to,an altered G/C content to more closely approach that typically found inthe plant species of interest, and the removal of codons atypicallyfound in the plant species commonly referred to as codon optimization.

The phrase “codon optimization” refers to the selection of appropriateDNA nucleotides for use within a structural gene or fragment thereofthat approaches codon usage within the plant of interest. Therefore, anoptimized gene or nucleic acid sequence refers to a gene in which thenucleotide sequence of a native or naturally occurring gene has beenmodified in order to utilize statistically-preferred orstatistically-favored codons within the plant. The nucleotide sequencetypically is examined at the DNA level and the coding region optimizedfor expression in the plant species determined using any suitableprocedure, for example as described in Sardana et al. (1996, Plant CellReports 15:677-681). In this method, the standard deviation of codonusage, a measure of codon usage bias, may be calculated by first findingthe squared proportional deviation of usage of each codon of the nativegene relative to that of highly expressed plant genes, followed by acalculation of the average squared deviation. The formula used is: 1SDCU=n=1N [(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage ofcodon n in highly expressed plant genes, where Yn to the frequency ofusage of codon n in the gene of interest and N refers to the totalnumber of codons in the gene of interest. A table of codon usage fromhighly expressed genes of dicotyledonous plants is compiled using thedata of Murray et al. (1989, Nuc Acids Res. 17:477-498).

One method of optimizing the nucleic acid sequence in accordance withthe preferred codon usage for a particular plant cell type is based onthe direct use, without performing any extra statistical calculations,of codon optimization tables such as those provided on-line at the CodonUsage Database through the NIAS (National Institute of AgrobiologicalSciences) DNA bank in Japan (wwwdotkazusadotordotjp/codon/). The CodonUsage Database contains codon usage tables for a number of differentspecies, with each codon usage table having been statisticallydetermined based on the data present in Genbank.

By using the above tables to determine the most preferred or mostfavored codons for each amino acid in a particular species (for example,rice), a naturally-occurring nucleotide sequence encoding a protein ofinterest can be codon optimized for that particular plant species. Thisis effected by replacing codons that may have a low statisticalincidence in the particular species genome with corresponding codons, inregard to an amino acid, that are statistically more favored. However,one or more less-favored codons may be selected to delete existingrestriction sites, to create new ones at potentially useful junctions(5′ and 3′ ends to add signal peptide or termination cassettes, internalsites that might be used to cut and splice segments together to producea correct full-length sequence), or to eliminate nucleotide sequencesthat may negatively effect mRNA stability or expression.

The naturally-occurring encoding nucleotide sequence may already, inadvance of any modification, contain a number of codons that correspondto a statistically-favored codon in a particular plant species.Therefore, codon optimization of the native nucleotide sequence maycomprise determining which codons, within the native nucleotidesequence, are not statistically-favored with regards to a particularplant, and modifying these codons in accordance with a codon usage tableof the particular plant to produce a codon optimized derivative. Amodified nucleotide sequence may be fully or partially optimized forplant codon usage provided that the protein encoded by the modifiednucleotide sequence is produced at a level higher than the proteinencoded by the corresponding naturally occurring or native gene.Construction of synthetic genes by altering the codon usage is describedin for example PCT Patent Application 93/07278.

As used herein, the phrase “microRNA (also referred to hereininterchangeably as “miRNA” or “miR”) or a precursor thereof” refers to amicroRNA (miRNA) molecule acting as a post-transcriptional regulator.Typically, the miRNA molecules are RNA molecules of about 20 to 22nucleotides in length which can be loaded into a RISC complex and whichdirect the cleavage of another RNA molecule, wherein the other RNAmolecule comprises a nucleotide sequence essentially complementary tothe nucleotide sequence of the miRNA molecule.

Typically, a miRNA molecule is processed from a “pre-miRNA” or as usedherein a precursor of a pre-miRNA molecule by proteins, such as DCLproteins, present in any plant cell and loaded onto a RISC complex whereit can guide the cleavage of the target RNA molecules.

Pre-microRNA molecules are typically processed from pri-microRNAmolecules (primary transcripts). The single stranded RNA segmentsflanking the pre-microRNA are important for processing of the pri-miRNAinto the pre-miRNA. The cleavage site appears to be determined by thedistance from the stem-ssRNA junction (Han et al. 2006, Cell 125,887-901, 887-901).

As used herein, a “pre-miRNA” molecule is an RNA molecule of about 100to about 200 nucleotides, preferably about 100 to about 130 nucleotideswhich can adopt a secondary structure comprising a double stranded RNAstem and a single stranded RNA loop (also referred to as “hairpin”) andfurther comprising the nucleotide sequence of the miRNA (and itscomplement sequence) in the double stranded RNA stem. According to aspecific embodiment, the miRNA and its complement are located about 10to about 20 nucleotides from the free ends of the miRNA double strandedRNA stem. The length and sequence of the single stranded loop region arenot critical and may vary considerably, e.g. between 30 and 50 nt inlength. The complementarity between the miRNA and its complement neednot be perfect and about 1 to 3 bulges of unpaired nucleotides can betolerated. The secondary structure adopted by an RNA molecule can bepredicted by computer algorithms conventional in the art such as mFOLD.The particular strand of the double stranded RNA stem from the pre-miRNAwhich is released by DCL activity and loaded onto the RISC complex isdetermined by the degree of complementarity at the 5′ end, whereby thestrand which at its 5′ end is the least involved in hydrogen boundingbetween the nucleotides of the different strands of the cleaved dsRNAstem is loaded onto the RISC complex and will determine the sequencespecificity of the target RNA molecule degradation. However, ifempirically the miRNA molecule from a particular synthetic pre-miRNAmolecule is not functional (because the “wrong” strand is loaded on theRISC complex), it will be immediately evident that this problem can besolved by exchanging the position of the miRNA molecule and itscomplement on the respective strands of the dsRNA stem of the pre-miRNAmolecule. As is known in the art, binding between A and U involving twohydrogen bounds, or G and U involving two hydrogen bounds is less strongthat between G and C involving three hydrogen bounds. Exemplary hairpinsequences are provided in Table 1, below.

According to the present teachings, the miRNA molecules may be naturallyoccurring or synthetic.

Naturally occurring miRNA molecules may be comprised within theirnaturally occurring pre-miRNA molecules but they can also be introducedinto existing pre-miRNA molecule scaffolds by exchanging the nucleotidesequence of the miRNA molecule normally processed from such existingpre-miRNA molecule for the nucleotide sequence of another miRNA ofinterest. The scaffold of the pre-miRNA can also be completelysynthetic. Likewise, synthetic miRNA molecules may be comprised within,and processed from, existing pre-miRNA molecule scaffolds or syntheticpre-miRNA scaffolds. Some pre-miRNA scaffolds may be preferred overothers for their efficiency to be correctly processed into the designedmicroRNAs, particularly when expressed as a chimeric gene wherein otherDNA regions, such as untranslated leader sequences or transcriptiontermination and polyadenylation regions are incorporated in the primarytranscript in addition to the pre-microRNA. One of ordinary skill in theart can decide on the proper pre-miRNA scaffold to use for generation ofthe miRNA molecules of the present invention while taking into accountthe presence of additional sequences which may influence the folding ofthe primary transcript RNA molecule into a secondary RNA structure andparticularly on presence and location of bulges or single stranded RNAstructures in otherwise doublestranded RNA stem (sub)structures. Thelocation of single-stranded RNA or bulge structures relative to thepre-miRNA, i.e. the distance in nucleotides should be carefullymaintained. Secondary RNA structures for a particular RNA nucleotidesequence can easily be predicted using software tools and algorithmswell known in the art such as mFOLD (Zucker et al. 2003 Nucleic AcidsResearch 31, 3406-3415). Furthermore, it is well within the skill of oneof ordinary skill in the art to design or modify a nucleotide bysubstituting nucleotides in a nucleotide sequence such that the newlyintroduced nucleotides exhibit more or less complementarity to anotherpart of the nucleotide sequence and in this way influence the generationof double-stranded RNA stems or of single stranded RNA bulges.

Thus, as mentioned the present invention envisages increasing toleranceof a plant to an abiotic stress or increasing biomass, vigor or yield ofa plant by upregulating (e.g., expressing), within the plant apolynucleotide of a microRNA selected from the group consisting ofmiR-156 (e.g. bna-miR156a, smo-miR156c, sbi-miR156d, smo-miR156d,vvi-miR156e, ath-miR156g, ptc-miR156k, zma-miR156k and osa-miR156l),miR-169 (e.g. ath-miR169a, osa-miR169a, sbi-miR169b, bna-miR169c,sbi-miR169c, ath-miR169d, osa-miR169e, bna-miR169g, sbi-miR169i,bna-miR169m, vvi-miR169m, ptc-miR169o, ptc-miR169q, ptc-miR169v andptc-miR169x), miR-164 (e.g. osa-miR164a, sbi-miR164b, osa-miR164c,osa-miR164e and ptc-miR164f), miR-159 (e.g. pta-miR159c, sof-miR159c,osa-miR159c and osa-miR159d), miR-167 (e.g. ppt-miR167, bna-miR167a,ath-miR167c, ath-miR167d, ptc-miR167f and ptc-miR167h), miR-529 (e.g.ppt-miR529a, ppt-miR529d, ppt-miR529e and ppt-miR529g), miR-168 (e.g.sbi-miR168 and gma-miR168), ppt-miR395, sof-miR408a, ath-miR408,miR-1039 (e.g. ppt-miR1039-3p), miR-1091 (e.g. smo-miR1091), miR-1118(e.g. tae-miR1118), miR-1134 (e.g. tae-miR1134) and miR-1129 (e.g.tae-miR1129). For the complete list of miRNAs contemplated by thepresent teachings, hairpins thereof and their corresponding sequencessee Table 1, below.

TABLE 1 Sequence identification of miRNAs and their correspondinghairpins hairpins MicroRNA miR SEQ ID NO. SEQ ID NO. osa-miR169e 1 11ath-miR169a 2 12, 194 ptc-miR169o 3 13 bna-miR169c 4 14 ptc-miR169v 5 15ath-miR169d 6 16 ath-miR167c 7 17 bna-miR167a 8 18 ppt-miR167 9 19ptc-miR167f 10 20 ppt-miR894 21 26 osa-miR164e 22 27 bna-miR169g 23 28vvi-miR169m 24 29 ptc-miR169q 25 30 bna-miR156a 31 49 ath-miR156g 32 50osa-miR156l 33 51 ath-miR854a 34 52 ppt-miR1039-3p 35 53 zma-miR156k 3654 vvi-miR156e 37 55 sbi-miR156d 38 56 ath-miR167d 39 57 ptc-miR167h 4058 sbi-miR168 41 59 gma-miR168 42 60 sof-miR408a 43 61 ath-miR408 44 62ppt-miR160b 45 63 ppt-miR160c 46 64 osa-miR390 47 65 ppt-miR390c 48 66sbi-miR172e 73 67 smo-miR171a 74 68 smo-miR171b 75 69 sbi-miR171b 76 70zma-miR171c 77 71 zma-miR171f 78 72 smo-miR156c 79 137 smo-miR156d 80138 ppt-miR395 81 139 smo-miR1091 82 140 tae-miR1118 83 141 tae-miR113484 142 vvi-miR171a 85 143 vvi-miR171i 86 144 gma-miR172a 87 145ath-miR172c 88 146 zma-miR172e 89 147 sbi-miR399b 90 148 osa-miR530-3p91 149 ppt-miR529a 92 150 ppt-miR529d 93 151 ppt-miR529e 94 152ppt-miR529g 95 153 osa-miR169a 96 154 sbi-miR169b 97 155 bna-miR169m 98156 ath-miR395a 99 157 bna-miR397a 100 158 ptc-miR397b 101 159smo-miR408 102 160 osa-miR528 103 161 ath-miR171b 104 162 ppt-miR896 105163 pta-miR159c 106 164 sof-miR159c 107 165 osa-miR159c 108 166osa-miR159d 109 167 osa-miR164a 110 168 sbi-miR164b 111 169 osa-miR164c112 170 ptc-miR164f 113 171 tae-miR1129 114 172 sof-miR168b 115 173osa-miR168b 116 174 sbi-miR169c 117 175 sbi-miR169i 118 176 ptc-miR169x119 177 sbi-miR171e 120 178 sbi-miR171f 121 179 mtr-miR399d 122 180ppt-miR477a-3p 123 181 ath-miR855 124 182 ppt-miR1026a 125 183ppt-miR901 126 184 sbi-miR166e 127 185 sbi-miR399a 128 186 ptc-miR156k129 187 osa-miR529b 130 188 vvi-miR535a 131 189 ptc-miR169t 132 190ath-miR156a 133 191 ath-miR164a 134 192 ath-miR167a 135 193

The present invention envisages the use of homologous sequences of theabove miRNAs. Thus, used are also nucleic acid sequences which are atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95% or more identical or similar to SEQ ID NOs: 1-193.Parameters for determining the level of identity are providedhereinbelow.

The miRNA molecules (or pre-miRNA processed into miRNA molecules) of thepresent teachings may alter the level of expression of the target genes(e.g. upregulate or downregulate) and consequently increase tolerance ofplants to severe stress (e.g. abiotic stress conditions) or increasebiomass, vigor or yield of the plant. Thus, the microRNAs of the presentteachings may bind, attach, regulate, process, interfere, and/ordestabilize any microRNA target. Such a target can be any molecule,including, but not limited to, DNA molecules, RNA molecules andpolypeptides.

As used herein a “target gene” refers to a gene that is processed bymicroRNA activity. Typically the gene encodes a polypeptide whichexpression is downregulated due to microRNA processing, however, thepresent invention also envisages target genes which have mRNA expressionproducts but not polypeptide products.

Thus as mentioned hereinabove, increasing tolerance of a plant to anabiotic stress or increasing biomass, vigor or yield of a plant, iseffected by expressing within the plant an exogenous polynucleotideencoding a nucleic acid agent capable of downregulating expression of atarget gene of a microRNA or a precursor thereof, wherein the microRNAis selected from the group consisting of miR-156, miR-169, miR-164,miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408,miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129, thereby increasingthe tolerance of the plant to the abiotic stress or increasing thebiomass, vigor or yield of the plant.

Target genes which are contemplated according to the present teachingsare provided in the polynucleotide sequences which comprise nucleic acidsequences as set forth in SEQ ID NO: 195-341 and 474-485. However thepresent teachings also relate to orthologs or homologs at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at leastabout 95% or more identical or similar to SEQ ID NO: 195-341 and474-485. Parameters for determining the level of identity are providedhereinbelow

Alternatively or additionally, target genes which are contemplatedaccording to the present teachings are provided in the polypeptidesequences which comprise amino acid sequences as set forth in SEQ ID NO:342-473 and 486-496. However the present teachings also relate to oforthologs or homologs at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, or at least about 95% or more identical or similarto SEQ ID NO: 342-473 and 486-496. Parameters for determining the levelof identity are provided hereinbelow

Examples of polynucleotide and polypeptide downregulating agents thatinhibit (also referred to herein as inhibitors) the expression of atarget gene are given below.

1. Polynucleotide-Based Inhibition of Gene Expression.

It will be appreciated, that any of these methods when specificallyreferring to downregulating expression/activity of the target genes canbe used, at least in part, to downregulate expression or activity ofendogenous miRNA molecules.

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression oftarget gene may be obtained by sense suppression or cosuppression. Forcosuppression, an expression cassette is designed to express an RNAmolecule corresponding to all or part of a messenger RNA encoding atarget gene in the “sense” orientation. Over-expression of the RNAmolecule can result in reduced expression of the native gene.Accordingly, multiple plant lines transformed with the cosuppressionexpression cassette are screened to identify those that show thegreatest inhibition of target gene expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the target gene, all or part of the 5′ and/or3′ untranslated region of a target transcript, or all or part of boththe coding sequence and the untranslated regions of a transcriptencoding the target gene. In some embodiments where the polynucleotidecomprises all or part of the coding region for the target gene, theexpression cassette is designed to eliminate the start codon of thepolynucleotide so that no protein product will be transcribed.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin, et al., (2002) PlantCell 15:1517-1532. Cosuppression may also be used to inhibit theexpression of multiple proteins in the same plant. Methods for usingcosuppression to inhibit the expression of endogenous genes in plantsare described in Flavell, et al., (1995) Proc. Natl. Acad. Sci. USA91:3590-3596; Jorgensen, et al., (1996) Plant Mol. Biol. 31:957-973;Johansen and Carrington, (2001) Plant Physiol. 126:930-938; Broin, etal., (2002) Plant Cell 15:1517-1532; Stoutjesdijk, et al., (2002) PlantPhysiol. 129:1723-1731; Yu, et al., (2003) Phytochemistry 63:753-763;and U.S. Pat. Nos. 5,035,323, 5,283,185 and 5,952,657; each of which isherein incorporated by reference. The efficiency of cosuppression may beincreased by including a poly-dt region in the expression cassette at aposition 3′ to the sense sequence and 5′ of the polyadenylation signal.See, US Patent Publication Number 20020058815, herein incorporated byreference. Typically, such a nucleotide sequence has substantialsequence identity to the sequence of the transcript of the endogenousgene, optimally greater than about 65% sequence identity, more optimallygreater than about 85% sequence identity, most optimally greater thanabout 95% sequence identity. See, U.S. Pat. Nos. 5,283,185 and5,035,323; herein incorporated by reference.

Transcriptional gene silencing (TGS) may be accomplished through use ofhpRNA constructs wherein the inverted repeat of the hairpin sharessequence identity with the promoter region of a gene to be silenced.Processing of the hpRNA into short RNAs which can interact with thehomologous promoter region may trigger degradation or methylation toresult in silencing. (Aufsatz, et al., (2002) PNAS 99(4):16499-16506;Mette, et al., (2000) EMBO J. 19(19):5194-5201)

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofthe target gene may be obtained by antisense suppression. For antisensesuppression, the expression cassette is designed to express an RNAmolecule complementary to all or part of a messenger RNA encoding thetarget gene. Over-expression of the antisense RNA molecule can result inreduced expression of the native gene. Accordingly, multiple plant linestransformed with the antisense suppression expression cassette arescreened to identify those that show the greatest inhibition of targetgene expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the target gene,all or part of the complement of the 5′ and/or 3′ untranslated region ofthe target gene transcript, or all or part of the complement of both thecoding sequence and the untranslated regions of a transcript encodingthe target gene. In addition, the antisense polynucleotide may be fullycomplementary (i.e., 100% identical to the complement of the targetsequence) or partially complementary (i.e., less than 100% identical tothe complement of the target sequence) to the target sequence. Antisensesuppression may be used to inhibit the expression of multiple proteinsin the same plant. Furthermore, portions of the antisense nucleotidesmay be used to disrupt the expression of the target gene. Generally,sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides,300, 500, 550, 500, 550 or greater may be used. Methods for usingantisense suppression to inhibit the expression of endogenous genes inplants are described, for example, in Liu, et al., (2002) Plant Physiol.129:1732-1753 and U.S. Pat. No. 5,759,829, which is herein incorporatedby reference. Efficiency of antisense suppression may be increased byincluding a poly-dt region in the expression cassette at a position 3′to the antisense sequence and 5′ of the polyadenylation signal. See, USPatent Publication Number 20020058815.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of atarget gene may be obtained by double-stranded RNA (dsRNA) interference.For dsRNA interference, a sense RNA molecule like that described abovefor cosuppression and an antisense RNA molecule that is fully orpartially complementary to the sense RNA molecule are expressed in thesame cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of target gene expression. Methods for usingdsRNA interference to inhibit the expression of endogenous plant genesare described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA95:13959-13965, Liu, et al., (2002) Plant Physiol. 129:1732-1753, and WO99/59029, WO 99/53050, WO 99/61631, and WO 00/59035;

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNAInterference

In some embodiments of the invention, inhibition of the expression ofone or more target gene may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at downregulating the expression ofendogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet.5:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited, and an antisense sequence that is fullyor partially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants. See, for example, Chuangand Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:5985-5990;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; andWaterhouse and Helliwell, (2003) Nat. Rev. Genet. 5:29-38. Methods forusing hpRNA interference to inhibit or silence the expression of genesare described, for example, in Chuang and Meyerowitz, (2000) Proc. Natl.Acad. Sci. USA 97:5985-5990; Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.5:29-38; Pandolfini, et al., BMC Biotechnology 3:7, and US PatentPublication Number 20030175965; each of which is herein incorporated byreference. A transient assay for the efficiency of hpRNA constructs tosilence gene expression in vivo has been described by Panstruga, et al.,(2003) Mol. Biol. Rep. 30:135-150, herein incorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith, et al., (2000) Nature507:319-320. In fact, Smith, et al., show 100% suppression of endogenousgene expression using ihpRNA-mediated interference. Methods for usingihpRNA interference to inhibit the expression of endogenous plant genesare described, for example, in Smith, et al., (2000) Nature 507:319-320;Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)Curr. Opin. Plant Biol. 5:156-150; Waterhouse and Helliwell, (2003) Nat.Rev. Genet. 5:29-38; Helliwell and Waterhouse, (2003) Methods30:289-295, and US Patent Publication Number 20030180955, each of whichis herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 02/00905, herein incorporated byreference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for target gene). Methods of usingamplicons to inhibit the expression of endogenous plant genes aredescribed, for example, in Angell and Baulcombe, (1997) EMBO J.16:3675-3685, Angell and Baulcombe, (1999) Plant J. 20:357-362, and U.S.Pat. No. 6,656,805, each of which is herein incorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of target gene. Thus, the polynucleotidecauses the degradation of the endogenous messenger RNA, resulting inreduced expression of the target gene. This method is described, forexample, in U.S. Pat. No. 5,987,071, herein incorporated by reference.

2. Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding a target polypeptide, resulting indownregulated expression of the gene. In particular embodiments, thezinc finger protein binds to a regulatory region of a miRNA or a targetpolypeptide. In other embodiments, the zinc finger protein binds to amessenger RNA encoding a miRNA or a target polypeptide and prevents itstranslation. Methods of selecting sites for targeting by zinc fingerproteins have been described, for example, in U.S. Pat. No. 6,553,252,and methods for using zinc finger proteins to inhibit the expression ofgenes in plants are described, for example, in US Patent PublicationNumber 20030037355; each of which is herein incorporated by reference.

3. Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one target polypeptide, anddownregulates the response regulator activity of the target polypeptide.In another embodiment, the binding of the antibody results in increasedturnover of the antibody-target polypeptide complex by cellular qualitycontrol mechanisms. The expression of antibodies in plant cells and theinhibition of molecular pathways by expression and binding of antibodiesto proteins in plant cells are well known in the art. See, for example,Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36, incorporatedherein by reference.

4. Gene Disruption

In some embodiments of the present invention, the activity of a miRNA ora target gene is reduced or eliminated by disrupting the gene encodingthe target polypeptide. The gene encoding the target polypeptide may bedisrupted by any method known in the art. For example, in oneembodiment, the gene is disrupted by transposon tagging. In anotherembodiment, the gene is disrupted by mutagenizing plants using random ortargeted mutagenesis, and selecting for plants that have reducedresponse regulator activity.

As mentioned, the present inventor has uncovered that increasingtolerance of a plant to an abiotic stress or increasing biomass, vigoror yield of a plant can be achieved by downregulating the activity orexpression of a miRNA selected from the group consisting of miR-171(e.g. smo-miR171a, vvi-miR171a, ath-miR171b, sbi-miR171b, smo-miR171b,zma-miR171c, sbi-miR171e, sbi-miR171f, zma-miR171f and vvi-miR171i),miR-172 (e.g. gma-miR172a, ath-miR172c and zma-miR172e), miR-399 (e.g.sbi-miR399a, sbi-miR399b and mtr-miR399d), miR-854 (e.g. ath-miR854a),miR-894, miR-160 (e.g. ppt-miR160b and ppt-miR160c), miR-166 (e.g.sbi-miR166e), miR-390 (e.g. osa-miR390 and ppt-miR390c), ath-miR395a,smo-miR408, miR-397 (e.g. bna-miR397a and ptc-miR397b), miR-477 (e.g.ppt-miR477a-3p), miR-528 (e.g. osa-miR528), miR-530 (e.g.osa-miR530-3p), miR-535 (e.g. vvi-miR535a), miR-855 (e.g. ath-miR855),miR-894 (e.g. ppt-miR894), miR-896 (e.g. ppt-miR896), miR-901 (e.g.ppt-miR901) and miR-1026 (e.g. ppt-miR1026a For the complete list ofmiRNAs contemplated by the present teachings, pre-miRNAs thereof andtheir corresponding sequences see Table 1, above.

Rendering miRNA molecules less functional or non-functional may beachieved in several ways as discussed in detail above.

Alternatively, downregulating the activity of miRNA molecules can beeffected by upregulating the expression of the target gene RNA (or atleast the part thereof recognized by the miRNA). Such an increase intarget gene RNA may be conveniently achieved by introducing into theplant cells a nucleic acid sequence encoding a polypeptide being atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, or atleast about 95% or more similar or identical to SEQ ID NO: 342-473 and486-496 under the regulation of a plant promoter.

Alternatively, such an increase in target gene RNA may be achieved byintroducing into the plant cells a nucleic acid sequence at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at leastabout 95% or more similar or identical to SEQ ID NOs: 195-341 and474-485 under the regulation of a plant promoter.

Identity (e.g., percent identity) can be determined using any homologycomparison software, including for example, the Basic Local AlignmentSearch Tool BlastN® (National Library of Medicine) software of theNational Center of Biotechnology Information (NCBI) such as by usingdefault parameters.

Homology (e.g., percent homology) can be determined using any homologycomparison software, including for example, the BlastP® (NationalLibrary of Medicine) or TBLASTN® (National Library of Medicine) softwareof the National Center of Biotechnology Information (NCBI) such as byusing default parameters, when starting from a polypeptide sequence; orthe tBLASTX® (National Library of Medicine) algorithm (available via theNCBI) such as by using default parameters, which compares the six-frameconceptual translation products of a nucleotide query sequence (bothstrands) against a protein sequence database. Homologous sequencesinclude both orthologous and paralogous sequences.

Exemplary target genes of miRNAs which may be expressed in plant cellsinclude, but are not limited to, the target gene of miR-171, the targetgene of miR-172, the target gene of miR-399, the target gene of miR-854,the target gene of miR-894, the target gene of miR-160, the target geneof miR-166, the target gene of miR-390, the target gene of ath-miR395a,the target gene of smo-miR408, the target gene of miR-397, the targetgene of miR-477, the target gene of miR-528, the target gene of miR-530,the target gene of miR-535, the target gene of miR-855, the target geneof miR-894, the target gene of miR-896, the target gene of miR-901 andthe target gene of miR-1026 (SEQ ID NOs: 195-496).

According to another embodiment of the present invention, downregulatingthe activity of a miRNA is effected by introducing into the plant atarget mimic or a micro-RNA resistant target which is bound by the miRNAbut is not cleaved by the microRNA.

According to a specific embodiment, the target mimic or micro-RNAresistant target may also be linked to the promoter naturally associatedwith the pre-miRNA recognizing the target gene and introduced into theplant cell. In this way, the miRNA target mimic or micro-RNA resistanttarget RNA will be expressed under the same circumstances as the miRNAand the target mimic or micro-RNA resistant target RNA will substitutefor the non-target mimic/micro-RNA resistant target RNA degraded by themiRNA induced cleavage.

Thus, the target mimic or micro-RNA resistant target is essentiallycomplementary to the microRNA provided that one or more of followingmismatches are allowed:

(a) a mismatch between the nucleotide at the 5′ end of the microRNA andthe corresponding nucleotide sequence in the target mimic or micro-RNAresistant target;

(b) a mismatch between any one of the nucleotides in position 1 toposition 9 of the microRNA and the corresponding nucleotide sequence inthe target mimic or micro-RNA resistant target; or

(c) three mismatches between any one of the nucleotides in position 12to position 21 of the microRNA and the corresponding nucleotide sequencein the target mimic or micro-RNA resistant target provided that thereare no more than two consecutive mismatches.

The target mimic RNA is essentially similar to the target RNA modifiedto render it resistant to miRNA induced cleavage, e.g. by modifying thesequence thereof such that a variation is introduced in the nucleotideof the target sequence complementary to the nucleotides 10 or 11 of themiRNA resulting in a mismatch. Clearly if the target RNA is a RNA codingfor a protein any modification would need to be silent with regard tothe coding region or at least result in a substitution yielding afunctional protein.

Alternatively, a microRNA-resistant target may be implemented. Thus, asilent mutation may be introduced in the microRNA binding site of thetarget gene so that the DNA and resulting RNA sequences are changed in away that prevents microRNA binding, but the amino acid sequence of theprotein is unchanged. Thus, a new sequence can be synthesized instead ofthe existing binding site, in which the DNA sequence is changed, but thetranslated amino acid sequence is retained resulted in lack of miRNAbinding to its target.

Non-functional miRNA alleles or miRNA resistant target genes may also beintroduced by homologous recombination to substitute the miRNA encodingalleles or miRNA sensitive target genes.

Recombinant expression is effected by cloning the nucleic acid ofinterest (e.g., miRNA, target gene, silencing agent etc) into a nucleicacid expression construct under the expression of a plant promoter.

According to some embodiments of the invention, there is provided anucleic acid construct, comprising a polynucleotide at least about 80%,at least about 85%, at least about 90%, at least about 95% or morehomologous to a nucleic acid sequence selected from the group consistingof miR-156, miR-169, miR-164, miR-159, miR-167, miR-529, miR-168,ppt-miR395, sof-miR408a, ath-miR408, miR-1039, miR-1091, miR-1118,miR-1134 and miR-1129 or a precursor thereof, wherein the nucleic acidsequence is under a transcriptional control of at least one promotercapable of directing transcription of the polynucleotide in a host cell.

It will be appreciated that the pre-miRNA molecules or miRNA moleculesof the present invention can be introduced into a plant by providing acell of the plant with a polynucleotide sequence comprising aplant-expressible promoter operably linked to a DNA region, which whentranscribed yields the pre-miRNA or miRNA molecule (explained furtherbelow). The plant expressible promoter may be the promoter naturallyassociated with the pre-miRNA molecule or it may be a heterologouspromoter.

According to some embodiments of the invention, there is provided anucleic acid construct, comprising a polynucleotide at least about 80%,at least about 85%, at least about 90%, at least about 95% or morehomologous to a nucleic acid sequence selected from the group consistingof a target gene of miR-171, a target gene of miR-172, a target gene ofmiR-399, a target gene of miR-854, a target gene of miR-894, a targetgene of miR-160, a target gene of miR-166, a target gene of miR-390, atarget gene of ath-miR395a, a target gene of smo-miR408, a target geneof miR-397, a target gene of miR-477, a target gene of miR-528, a targetgene of miR-530, a target gene of miR-535, a target gene of miR-855, atarget gene of miR-894, a target gene of miR-896, a target gene ofmiR-901 and a target gene of miR-1026, wherein the nucleic acid sequenceis under a transcriptional control of at least one promoter capable ofdirecting transcription of the polynucleotide in a host cell.

According to some embodiments of the invention, there is provided anucleic acid construct, comprising a nucleic acid sequence fordown-regulating an expression of a target gene of a microRNA or aprecursor thereof, wherein the target gene of the microRNA is selectedfrom the group consisting of a target gene of miR-156, a target gene ofmiR-169, a target gene of miR-164, a target gene of miR-159, a targetgene of miR-167, a target gene of miR-529, a target gene of miR-168, atarget gene of ppt-miR395, a target gene of sof-miR408a, a target geneof ath-miR408, a target gene of miR-1039, a target gene of miR-1091, atarget gene of miR-1118, a target gene of miR-1134 and a target gene ofmiR-1129, wherein the nucleic acid sequence is under a transcriptionalcontrol of at least one promoter capable of directing transcription ofthe polynucleotide in a host cell.

According to a specific example of the present teachings, the targetgene of miR-169 is a NF-YA8 protein.

According to some embodiments of the invention, there is provided anucleic acid construct, comprising a nucleic acid sequence fordown-regulating an expression of a microRNA or a precursor thereof,wherein the microRNA is selected from the group consisting of miR-171,miR-172, miR-399, miR-854, miR-894, miR-160, miR-166, miR-390,ath-miR395a, smo-miR408, miR-397, miR-477, miR-528, miR-530, miR-535,miR-855, miR-894, miR-896, miR-901 and miR-1026, wherein the nucleicacid sequence is under a transcriptional control of at least onepromoter capable of directing transcription of the polynucleotide in ahost cell.

According to a specific embodiment, the host cell comprises a plantcell.

Expressing the exogenous polynucleotides of the present invention (e.g.miRNA molecules, targets thereof, mimic sequences, downregulatingagents) may be effected by transforming a cell of a plant with theexogenous polynucleotide.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The plantmay be in any form including suspension cultures, embryos, meristematicregions, callus tissue, leaves, gametophytes, sporophytes, pollen, andmicrospores.

As used herein the phrase “plant cell” refers to plant cells which arederived and isolated from disintegrated plant cell tissue or plant cellcultures.

As used herein the phrase “plant cell culture” refers to any type ofnative (naturally occurring) plant cells, plant cell lines andgenetically modified plant cells, which are not assembled to form acomplete plant, such that at least one biological structure of a plantis not present. Optionally, the plant cell culture of this aspect of thepresent invention may comprise a particular type of a plant cell or aplurality of different types of plant cells. It should be noted thatoptionally plant cultures featuring a particular type of plant cell maybe originally derived from a plurality of different types of such plantcells.

Any commercially or scientifically valuable plant is envisaged inaccordance with these embodiments of the invention. Plants that areparticularly useful in the methods of the invention include all plantswhich belong to the superfamily Viridiplantae, in particularmonocotyledonous and dicotyledonous plants including a fodder or foragelegume, ornamental plant, food crop, tree, or shrub selected from thelist comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp.,Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp.,Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaeaplurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkeaafricana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camelliasinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens,Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermummopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumisspp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeriajaponica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergiamonetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa,Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestisspp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulaliavi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingiaspp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffheliadissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago saliva, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara,Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysvefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize,wheat, barely, rye, oat, peanut, pea, lentil and alfalfa, cotton,rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, atree, an ornamental plant, a perennial grass and a forage crop.Alternatively algae and other non-Viridiplantae can be used for themethods of the present invention.

According to some embodiments of the invention, the plant used by themethod of the invention is a crop plant including, but not limited to,cotton, Brassica vegetables, oilseed rape, sesame, olive tree, palm oil,banana, wheat, corn or maize, barley, alfalfa, peanuts, sunflowers,rice, oats, sugarcane, soybean, turf grasses, barley, rye, sorghum,sugar cane, chicory, lettuce, tomato, zucchini, bell pepper, eggplant,cucumber, melon, watermelon, beans, hibiscus, okra, apple, rose,strawberry, chile, garlic, pea, lentil, canola, mums, arabidopsis,broccoli, cabbage, beet, quinoa, spinach, squash, onion, leek, tobacco,potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, andalso plants used in horticulture, floriculture or forestry, such as, butnot limited to, poplar, fir, eucalyptus, pine, an ornamental plant, aperennial grass and a forage crop, coniferous plants, moss, algae, aswell as other plants listed in World Wide Web (dot) nationmaster (dot)com/encyclopedia/Plantae.

According to a specific embodiment of the present invention, the plantcomprises corn.

According to a specific embodiment of the present invention, the plantcomprises sorghum.

According to some embodiments of the invention, there is provided aplant cell exogenously expressing the polynucleotide of some embodimentsof the invention, the nucleic acid construct of some embodiments of theinvention and/or the polypeptide of some embodiments of the invention.

According to some embodiments of the invention, expressing the exogenouspolynucleotide of the invention within the plant is effected bytransforming one or more cells of the plant with the exogenouspolynucleotide, followed by generating a mature plant from thetransformed cells and cultivating the mature plant under conditionssuitable for expressing the exogenous polynucleotide within the matureplant.

According to some embodiments of the invention, the transformation iseffected by introducing to the plant cell a nucleic acid construct whichincludes the exogenous polynucleotide of some embodiments of theinvention and at least one promoter capable of directing transcriptionof the exogenous polynucleotide in the plant cell. Further details ofsuitable transformation approaches are provided herein below.

Exemplary nucleic acid constructs which have been constructed and usedfor plant transformation by the present inventor include pORE156,pORE164, pORE167 and pORE169, which were all constructed by ligating theappropriate DNA fragments into the pORE E2 binary vector (Accessionnumber: AY562535) under the transcriptional control of a promoter. Thefollowing nucleic acid sequences where used, respectively, therein:ath-miR156a (SEQ ID NO: 191), ath-miR164a (SEQ ID NO: 192), ath-miR167a(SEQ ID NO: 193), and ath-miR169a (SEQ ID NO: 12), as described indetail in Example 5 of the Examples section which follows.

A coding nucleic acid sequence is “operably linked” to a regulatorysequence (e.g., promoter) if the regulatory sequence is capable ofexerting a regulatory effect on the coding sequence linked thereto.

As used herein, the term “promoter” denotes any DNA which is recognizedand bound (directly or indirectly) by a DNA-dependent RNA-polymeraseduring initiation of transcription. The promoter controls where (e.g.,which portion of a plant) and/or when (e.g., at which stage or conditionin the lifetime of an organism) the gene is expressed. Thus, a promoterincludes the transcription initiation site, and binding sites fortranscription initiation factors and RNA polymerase, and can comprisevarious other sites (e.g., enhancers), at which gene expressionregulatory proteins may bind.

The term “regulatory sequence”, as used herein, means any DNA, that isinvolved in driving transcription and controlling (i.e., regulating) thetiming and level of transcription of a given DNA sequence, such as a DNAcoding for a protein or polypeptide. For example, a 5′ regulatory region(or “promoter region”) is a DNA sequence located upstream (i.e., 5′) ofa coding sequence and which comprises the promoter and the5′-untranslated leader sequence. A 3′ regulatory region is a DNAsequence located downstream (i.e., 3′) of the coding sequence and whichcomprises suitable transcription termination (and/or regulation)signals, including one or more polyadenylation signals.

For the purpose of the invention, the promoter is a plant-expressiblepromoter. As used herein, the term “plant-expressible promoter” means aDNA sequence which is capable of controlling (initiating) transcriptionin a plant cell. This includes any promoter of plant origin, but alsoany promoter of non-plant origin which is capable of directingtranscription in a plant cell, i.e., certain promoters of viral orbacterial origin Thus, any suitable promoter sequence can be used by thenucleic acid construct of the present invention. According to someembodiments of the invention, the promoter is a constitutive promoter, atissue-specific promoter or an inducible promoter (e.g. an abioticstress-inducible promoter).

Suitable constitutive promoters include, for example, hydroperoxidelyase (HPL) promoter, CaMV 35S promoter (Odell et al, Nature313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No.WO04081173A2); Arabidopsis new At6669 promoter; maize Ubi 1 (Christensenet al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al.,Plant Cell 2:163-171, 1990); pEMU (Last et al, Theor. Appl. Genet.81:581-588, 1991); CaMV 19S (Nilsson et al, Physiol. Plant 100:456-462,1997); GOS2 (de Pater et al, Plant J Nov; 2(6):837-44, 1992); ubiquitin(Christensen et al, Plant MoI. Biol. 18: 675-689, 1992); Ricecyclophilin (Bucholz et al, Plant MoI Biol. 25(5):837-43, 1994); MaizeH3 histone (Lepetit et al, MoI. Gen. Genet. 231:276-285, 1992); Actin 2(An et al, Plant J. 10(1); 107-121, 1996) and Synthetic Super MAS (Ni etal., The Plant Journal 7: 661-76, 1995). Other constitutive promotersinclude those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5,608,144;5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Suitable tissue-specific promoters include, but not limited to,leaf-specific promoters [such as described, for example, by Yamamoto etal., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67,1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor etal., Plant J. 3:509-18, 1993; Orozco et al., Plant MoI. Biol.23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA90:9586-9590, 1993], seed-preferred promoters [e.g., from seed specificgenes (Simon, et al., Plant MoI. Biol. 5. 191, 1985; Scofield, et al.,J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant MoI. Biol.14: 633, 1990), Brazil Nut albumin (Pearson′ et al., Plant MoI. Biol.18: 235-245, 1992), legumin (Ellis, et al. Plant MoI. Biol. 10: 203-214,1988), Glutelin (rice) (Takaiwa, et al., MoI. Gen. Genet. 208: 15-22,1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke etal., Plant MoI Biol, 143)323-32 1990), napA (Stalberg, et al., Planta199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171-184,1997), sunflower oleosin (Cummins, et al, Plant MoI. Biol. 19: 873-876,1992)], endosperm specific promoters [e.g., wheat LMW and HMW,glutenin-1 (MoI Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b andg gliadins (EMBO3: 1409-15, 1984), Barley ltrl promoter, barley Bl, C, Dhordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; MoIGen Genet 250:750-60, 1996), Barley DOF (Mena et al., The Plant Journal,116(1): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter(Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolaminNRP33, rice-globulin GIb-I (Wu et al., Plant Cell Physiology 39(8)885-889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant MoI.Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68,1997), maize ESR gene family (Plant J 12:235-46, 1997), sorghumgamma-kafirin (PMB 32:1029-35, 1996); e.g., the Napin promoter], embryospecific promoters [e.g., rice OSH1 (Sato et al, Proc. Natl. Acad. Sci.USA, 93: 8117-8122), KNOX (Postma-Haarsma et al, Plant MoI. Biol.39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)],and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA)(Van der Meer, et al., Plant MoI. Biol. 15, 95-109, 1990), LAT52 (Twellet al., MoI. Gen Genet. 217:240-245; 1989), apetala-3].

Suitable abiotic stress-inducible promoters include, but not limited to,salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al.,MoI. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such asmaize rab17 gene promoter (PIa et al., Plant MoI. Biol. 21:259-266,1993), maize rab28 gene promoter (Busk et al., Plant J. 11:1285-1295,1997) and maize Ivr2 gene promoter (Pelleschi et al., Plant MoI. Biol.39:373-380, 1999); heat-inducible promoters such as heat tomatohsp80-promoter from tomato (U.S. Pat. No. 5,187,267).

The nucleic acid construct of some embodiments of the invention canfurther include an appropriate selectable marker and/or an origin ofreplication. According to some embodiments of the invention, the nucleicacid construct utilized is a shuttle vector, which can propagate both inE. coli (wherein the construct comprises an appropriate selectablemarker and origin of replication) and be compatible with propagation incells. The construct according to some embodiments of the invention canbe, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, avirus or an artificial chromosome.

The nucleic acid construct of some embodiments of the invention can beutilized to stably or transiently transform plant cells. In stabletransformation, the exogenous polynucleotide is integrated into theplant genome and as such it represents a stable and inherited trait. Intransient transformation, the exogenous polynucleotide is expressed bythe cell transformed but it is not integrated into the genome and assuch it represents a transient trait.

When naked RNA or DNA is introduced into a cell, the polynucleotides maybe synthesis using any method known in the art, including eitherenzymatic syntheses or solid-phase syntheses. These are especiallyuseful in the case of short polynucleotide sequences with or withoutmodifications as explained above. Equipment and reagents for executingsolid-phase synthesis are commercially available from, for example,Applied Biosystems. Any other means for such synthesis may also beemployed; the actual synthesis of the oligonucleotides is well withinthe capabilities of one skilled in the art and can be accomplished viaestablished methodologies as detailed in, for example: Sambrook, J. andRussell, D. W. (2001), “Molecular Cloning: A Laboratory Manual”;Ausubel, R. M. et al., eds. (1994, 1989), “Current Protocols inMolecular Biology,” Volumes I-III, John Wiley & Sons, Baltimore, Md.;Perbal, B. (1988), “A Practical Guide to Molecular Cloning,” John Wiley& Sons, New York; and Gait, M. J., ed. (1984), “OligonucleotideSynthesis”; utilizing solid-phase chemistry, e.g. cyanoethylphosphoramidite followed by deprotection, desalting, and purificationby, for example, an automated trityl-on method or HPLC.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, L, Annu. Rev.Plant. Physiol, Plant. MoI. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer (e.g., T-DNA usingAgrobacterium tumefaciens or Agrobacterium rhizogenes); see for example,Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogersin Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, MolecularBiology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K.,Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in PlantBiotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth Publishers,Boston, Mass. (1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. See, e.g., Horsch et al. in Plant Molecular BiologyManual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. Asupplementary approach employs the Agrobacterium delivery system incombination with vacuum infiltration. The Agrobacterium system isespecially viable in the creation of transgenic dicotyledonous plants.

According to a specific embodiment of the present invention, theexogenous polynucleotide is introduced into the plant by infecting theplant with a bacteria, such as using a floral dip transformation method(as described in further detail in Example 5, of the Examples sectionwhich follows).

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. For thisreason it is preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

Although stable transformation is presently preferred, transienttransformation of leaf cells, meristematic cells or the whole plant isalso envisaged by the present invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus(BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation ofplants using plant viruses is described in U.S. Pat. No. 4,855,237 (beangolden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese PublishedApplication No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); andGluzman, Y. et al., Communications in Molecular Biology: Viral Vectors,Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirusparticles for use in expressing foreign DNA in many hosts, includingplants are described in WO 87/06261. According to some embodiments ofthe invention, the virus used for transient transformations is avirulentand thus is incapable of causing severe symptoms such as reduced growthrate, mosaic, ring spots, leaf roll, yellowing, streaking, poxformation, tumor formation and pitting. A suitable avirulent virus maybe a naturally occurring avirulent virus or an artificially attenuatedvirus. Virus attenuation may be effected by using methods well known inthe art including, but not limited to, sub-lethal heating, chemicaltreatment or by directed mutagenesis techniques such as described, forexample, by Kurihara and Watanabe (Molecular Plant Pathology 4:259-269,2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as,for example, the American Type culture Collection (ATCC) or by isolationfrom infected plants. Isolation of viruses from infected plant tissuescan be effected by techniques well known in the art such as described,for example by Foster and Tatlor, Eds. “Plant Virology Protocols: FromVirus Isolation to Transgenic Resistance (Methods in Molecular Biology(Humana Pr), VoI 81)”, Humana Press, 1998. Briefly, tissues of aninfected plant believed to contain a high concentration of a suitablevirus, preferably young leaves and flower petals, are ground in a buffersolution (e.g., phosphate buffer solution) to produce a virus infectedsap which can be used in subsequent inoculations.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous polynucleotide sequences in plants is demonstratedby the above references as well as by Dawson, W. O. et al, Virology(1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French etal. Science (1986) 231:1294-1297; Takamatsu et al. FEBS Letters (1990)269:73-76; and U.S. Pat. No. 5,316,931.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinswhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native nucleic acid sequencewithin it, such that a protein is produced. The recombinant plant viralnucleic acid may contain one or more additional non-native subgenomicpromoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or nucleic acid sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) nucleic acid sequencesmay be inserted adjacent the native plant viral subgenomic promoter orthe native and a non-native plant viral subgenomic promoters if morethan one nucleic acid sequence is included. The non-native nucleic acidsequences are transcribed or expressed in the host plant under controlof the subgenomic promoter to produce the desired products.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that the sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(isolated nucleic acid) in the host to produce the desired protein.

In addition to the above, the nucleic acid molecule of the presentinvention can also be introduced into a chloroplast genome therebyenabling chloroplast expression.

A technique for introducing exogenous nucleic acid sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous nucleic acid is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous nucleic acidmolecule into the chloroplasts. The exogenous nucleic acid is selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the exogenous nucleic acid includes, inaddition to a gene of interest, at least one nucleic acid stretch whichis derived from the chloroplast's genome. In addition, the exogenousnucleic acid includes a selectable marker, which serves by sequentialselection procedures to ascertain that all or substantially all of thecopies of the chloroplast genomes following such selection will includethe exogenous nucleic acid. Further details relating to this techniqueare found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast and become integratedinto the chloroplast's inner membrane.

Since tolerance to abiotic stress as well as yield, vigor or biomass ofthe plant can involve multiple genes acting additively or in synergy(see, for example, in Quesda et al., Plant Physiol. 130:951-063, 2002),the invention also envisages expressing a plurality of exogenouspolynucleotides in a single host plant to thereby achieve superioreffect on tolerance to abiotic stress, yield, vigor and biomass of theplant.

Expressing a plurality of exogenous polynucleotides in a single hostplant can be effected by co-introducing multiple nucleic acidconstructs, each including a different exogenous polynucleotide, into asingle plant cell. The transformed cell can then be regenerated into amature plant using the methods described hereinabove. Alternatively,expressing a plurality of exogenous polynucleotides in a single hostplant can be effected by co-introducing into a single plant-cell asingle nucleic-acid construct including a plurality of differentexogenous polynucleotides. Such a construct can be designed with asingle promoter sequence which can transcribe a polycistronic messengerRNA including all the different exogenous polynucleotide sequences. Toenable co-translation of the different polypeptides encoded by thepolycistronic messenger RNA, the polynucleotide sequences can beinter-linked via an internal ribosome entry site (IRES) sequence whichfacilitates translation of polynucleotide sequences positioneddownstream of the IRES sequence. In this case, a transcribedpolycistronic RNA molecule encoding the different polypeptides describedabove will be translated from both the capped 5′ end and the twointernal IRES sequences of the polycistronic RNA molecule to therebyproduce in the cell all different polypeptides. Alternatively, theconstruct can include several promoter sequences each linked to adifferent exogenous polynucleotide sequence.

The plant cell transformed with the construct including a plurality ofdifferent exogenous polynucleotides can be regenerated into a matureplant, using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides canbe effected by introducing different nucleic acid constructs, includingdifferent exogenous polynucleotides, into a plurality of plants. Theregenerated transformed plants can then be cross-bred and resultantprogeny selected for superior yield (e.g., tolerance to abiotic stres),using conventional plant breeding techniques.

According to some embodiments of the invention, the plant expressing theexogenous polynucleotide(s) is grown under non-stress or normalconditions (e.g., biotic conditions and/or conditions with sufficientwater, nutrients such as nitrogen and fertilizer). Such conditions,which depend on the plant being grown, are known to those skilled in theart of agriculture, and are further, described above.

According to some embodiments of the invention, the method furthercomprises growing the plant expressing the exogenous polynucleotide(s)under abiotic stress. Non-limiting examples of abiotic stress conditionsinclude, water deprivation, drought, excess of water (e.g., flood,waterlogging), freezing, low temperature, high temperature, strongwinds, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrientexcess, salinity, atmospheric pollution, intense light, insufficientlight, or UV irradiation, etiolation and atmospheric pollution.

Thus, the invention encompasses plants exogenously expressing thepolynucleotide(s), the nucleic acid constructs and/or polypeptide(s) ofthe invention. Once expressed within the plant cell or the entire plant,the level of the polypeptide encoded by the exogenous polynucleotide canbe determined by methods well known in the art such as, activity assays,Western blots using antibodies capable of specifically binding thepolypeptide, Enzyme-Linked Immuno Sorbent Assay (ELISA),radio-immuno-assays (RIA), immunohistochemistry, immunocytochemistry,immunofluorescence and the like.

Methods of determining the level in the plant of the RNA transcribedfrom the exogenous polynucleotide are well known in the art and include,for example, Northern blot analysis, reverse transcription polymerasechain reaction (RT-PCR) analysis (including quantitative,semi-quantitative or real-time RT-PCR) and RNA-m situ hybridization.

The sequence information and annotations uncovered by the presentteachings can be harnessed in favor of classical breeding. Thus,sub-sequence data of those polynucleotides described above, can be usedas markers for marker assisted selection (MAS), in which a marker isused for indirect selection of a genetic determinant or determinants ofa trait of interest (e.g., tolerance to abiotic stress). Nucleic aciddata of the present teachings (DNA or RNA sequence) may contain or belinked to polymorphic sites or genetic markers on the genome such asrestriction fragment length polymorphism (RFLP), microsatellites andsingle nucleotide polymorphism (SNP), DNA fingerprinting (DFP),amplified fragment length polymorphism (AFLP), expression levelpolymorphism, polymorphism of the encoded polypeptide and any otherpolymorphism at the DNA or RNA sequence.

Examples of marker assisted selections include, but are not limited to,selection for a morphological trait (e.g., a gene that affects form,coloration, male sterility or resistance such as the presence or absenceof awn, leaf sheath coloration, height, grain color, aroma of rice);selection for a biochemical trait (e.g., a gene that encodes a proteinthat can be extracted and observed; for example, isozymes and storageproteins); selection for a biological trait (e.g., pathogen races orinsect biotypes based on host pathogen or host parasite interaction canbe used as a marker since the genetic constitution of an organism canaffect its susceptibility to pathogens or parasites).

The polynucleotides and polypeptides described hereinabove can be usedin a wide range of economical plants, in a safe and cost effectivemanner.

Plant lines exogenously expressing the polynucleotide or the polypeptideof the invention can be screened to identify those that show thegreatest increase of the desired plant trait.

Thus, according to an additional embodiment of the present invention,there is provided a method of evaluating a trait of a plant, the methodcomprising: (a) expressing in a plant or a portion thereof the nucleicacid construct; and (b) evaluating a trait of a plant as compared to awild type plant of the same type; thereby evaluating the trait of theplant.

Thus, the effect of the transgene (the exogenous polynucleotide encodingthe polypeptide) on different plant characteristics may be determinedany method known to one of ordinary skill in the art.

Thus, for example, tolerance to abiotic stress may be compared intransformed plants {i.e., expressing the transgene) compared tonon-transformed (wild type) plants exposed to the same abiotic stressconditions (e.g. water deprivation, salt stress e.g. salinity,suboptimal temperature, nutrient deficiency, nutrient excess, osmoticstress, and the like), using the following assays (also described inExamples 7 of the Examples section which follows):

Drought tolerance assay—Soil-based drought screens are performed withplants overexpressing the polynucleotides detailed above. Seeds fromcontrol Arabidopsis plants, or other transgenic plants overexpressingthe polypeptide of the invention are germinated and transferred to pots.Drought stress is obtained after irrigation is ceased. Transgenic andcontrol plants are compared to each other when the majority of thecontrol plants develop severe wilting. Plants are re-watered afterobtaining a significant fraction of the control plants displaying asevere wilting. Plants are ranked comparing to controls for each of twocriteria: tolerance to the drought conditions and recovery (survival)following re-watering.

Quantitative parameters of tolerance measured include, but are notlimited to, the average wet and dry weight, growth rate, leaf size, leafcoverage (overall leaf area), the weight of the seeds yielded, theaverage seed size and the number of seeds produced per plant.Transformed plants not exhibiting substantial physiological and/ormorphological effects, or exhibiting higher biomass than wild-typeplants, are identified as drought stress tolerant plants

Salinity tolerance assay—Transgenic plants with tolerance to high saltconcentrations are expected to exhibit better germination, seedlingvigor or growth in high salt. Salt stress can be effected in many wayssuch as, for example, by irrigating the plants with a hyperosmoticsolution, by cultivating the plants hydroponically in a hyperosmoticgrowth solution (e.g., Hoagland solution with added salt), or byculturing the plants in a hyperosmotic growth medium [e.g., 50%Murashige-Skoog medium (MS medium) with added salt]. Since differentplants vary considerably in their tolerance to salinity, the saltconcentration in the irrigation water, growth solution, or growth mediumcan be adjusted according to the specific characteristics of thespecific plant cultivar or variety, so as to inflict a mild or moderateeffect on the physiology and/or morphology of the plants (for guidelinesas to appropriate concentration see, Bernstein and Kafkafi, Root GrowthUnder Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y,Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002, andreference therein).

For example, a salinity tolerance test can be performed by irrigatingplants at different developmental stages with increasing concentrationsof sodium chloride (for example 50 mM, 150 mM, 300 mM NaCl) applied fromthe bottom and from above to ensure even dispersal of salt. Followingexposure to the stress condition the plants are frequently monitoreduntil substantial physiological and/or morphological effects appear inwild type plants. Thus, the external phenotypic appearance, degree ofchlorosis and overall success to reach maturity and yield progeny arecompared between control and transgenic plants. Quantitative parametersof tolerance measured include, but are not limited to, the average wetand dry weight, growth rate, leaf size, leaf coverage (overall leafarea), the weight of the seeds yielded, the average seed size and thenumber of seeds produced per plant. Transformed plants not exhibitingsubstantial physiological and/or morphological effects, or exhibitinghigher biomass than wild-type plants, are identified as abiotic stresstolerant plants.

Osmotic tolerance test—Osmotic stress assays (including sodium chlorideand PEG assays) are conducted to determine if an osmotic stressphenotype was sodium chloride-specific or if it was a general osmoticstress related phenotype. Plants which are tolerant to osmotic stressmay have more tolerance to drought and/or freezing. For salt and osmoticstress experiments, the medium is supplemented for example with 50 mM,100 mM, 200 mM NaCl or 15%, 20% or 25% PEG.

Cold stress tolerance—One way to analyze cold stress is as follows.Mature (25 day old) plants are transferred to 4° C. chambers for 1 or 2weeks, with constitutive light. Later on plants are moved back togreenhouse. Two weeks later damages from chilling period, resulting ingrowth retardation and other phenotypes, are compared between controland transgenic plants, by measuring plant weight (wet and dry), and bycomparing growth rates measured as time to flowering, plant size, yield,and the like.

Heat stress tolerance—One way to measure heat stress tolerance is byexposing the plants to temperatures above 34° C. for a certain period.Plant tolerance is examined after transferring the plants back to 22° C.for recovery and evaluation after 5 days relative to internal controls(non-transgenic plants) or plants not exposed to neither cold or heatstress.

The biomass, vigor and yield of the plant can also be evaluated usingany method known to one of ordinary skill in the art. Thus, for example,plant vigor can be calculated by the increase in growth parameters suchas leaf area, fiber length, rosette diameter, plant fresh weight and thelike per time.

As mentioned, the increase of plant yield can be determined by variousparameters. For example, increased yield of rice may be manifested by anincrease in one or more of the following: number of plants per growingarea, number of panicles per plant, number of spikelets per panicle,number of flowers per panicle, increase in the seed filling rate,increase in thousand kernel weight (1000-weight), increase oil contentper seed, increase starch content per seed, among others. An increase inyield may also result in modified architecture, or may occur because ofmodified architecture. Similarly, increased yield of soybean may bemanifested by an increase in one or more of the following: number ofplants per growing area, number of pods per plant, number of seeds perpod, increase in the seed filling rate, increase in thousand seed weight(1000-weight), reduce pod shattering, increase oil content per seed,increase protein content per seed, among others. An increase in yieldmay also result in modified architecture, or may occur because ofmodified architecture.

Thus, the present invention is of high agricultural value for increasingtolerance of plants to abiotic stress as well as promoting the yield,biomass and vigor of commercially desired crops.

According to another embodiment of the present invention, there isprovided a food or feed comprising the plants or a portion thereof ofthe present invention.

In a further aspect the invention, the transgenic plants of the presentinvention or parts thereof are comprised in a food or feed product(e.g., dry, liquid, paste). A food or feed product is any ingestiblepreparation containing the transgenic plants, or parts thereof, of thepresent invention, or preparations made from these plants. Thus, theplants or preparations are suitable for human (or animal) consumption,i.e. the transgenic plants or parts thereof are more readily digested.Feed products of the present invention further include a oil or abeverage adapted for animal consumption.

It will be appreciated that the transgenic plants, or parts thereof, ofthe present invention may be used directly as feed products oralternatively may be incorporated or mixed with feed products forconsumption. Furthermore, the food or feed products may be processed orused as is. Exemplary feed products comprising the transgenic plants, orparts thereof, include, but are not limited to, grains, cereals, such asoats, e.g. black oats, barley, wheat, rye, sorghum, corn, vegetables,leguminous plants, especially soybeans, root vegetables and cabbage, orgreen forage, such as grass or hay.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

General Materials and Experimental Procedures

Plant Material

Corn seeds were obtained from Galil seeds (Israel). Corn variety 5605was used in all experiments. Plants were grown at 28° C. under a 16hours light:8 hours dark regime.

Stress Induction

Plants were grown under standard conditions as described above untilseedlings were two weeks old. Next, plants were divided into threegroups: control plants were irrigated with tap water twice weekly,salinity-treated plants were irrigated with tap water spiked with 150 or300 mM NaCl, as specified in Tables 2-5 (below), twice weekly, anddrought-treated plants received no irrigation. The experiment continuedas described in the specific examples, after which plants were harvestedfor RNA extraction.

Total RNA Extraction

Total RNA of leaf or root samples from five to six biological repeatswere extracted using the mirVana™ kit (Ambion, Austin, Tex.) as follows:

Samples were ground with liquid nitrogen and the resulting powder(300-400 mg) was placed in a 13-ml tube. Lysis buffer (2 ml) andhomogenate additive (200 μl) were added, mixed well and incubated for 10min on ice. Samples were extracted twice with a volume of acidphenol-chloroform (2 ml), vortexed for 30-60 s and centrifuged for 5 minat 12,000 g. Samples were further extracted with one volume ofchloroform:isoamyl 24:1, and the aqueous phase was transferred to afresh 13-ml tube. Ethanol (100%, 1.25 volumes) was added to the aqueousphase and the mixture was loaded onto a column in 700 μl aliquots.

The column was washed once with Wash Solution 1 (650 μl) and twice morewith Wash Solution 2/3 (500 μl). Next, the column was centrifuged for 1min to remove residual fluid and the column was transferred to a newcollection tube. RNAse-free water (100 μl), pre-heated to 95° C., wasadded to the column and the column was centrifuged for 1 min at 14,000rpm. The elution step was performed twice with the same water.

RNA concentration was measured using an ND-1000 Spectrophotometer(NanoDrop, USA). DNase Turbo® (Ambion Inc., USA) was added at 1 μlenzyme per 10 μg RNA and the mixture was incubated for 60 min at 37° C.An equal volume of acid phenol-chloroform was added, and the sampleswere vortexed and centrifuged for 10 min at 12,000 g. The upper phasewas transferred to a new tube, and a 10% volume of NaOAc 3 M, pH 5.2,was added, followed by three volumes of 100% ethanol. Tubes wereincubated overnight at −20° C. and precipitated by centrifugation at 4°C. for 40 min at 14,000 rpm. The supernatant was removed and washed with0.5 ml of 85% cold ethanol. The pellet was dried and re-suspended in 50μl of RNAse-free water.

Database

MicroRNA sequences were derived from versions 10.1 and 14 of miRBase(released December 2007) and comprise the 673 non-redundant sequencesdefined as “Viridiplantae” and the 47 sequences submitted for C.reinhardtii.

Microarray Design

Custom microarrays were manufactured by Agilent Technologies by in situsynthesis of DNA oligonucleotide probes for 890 plant and algalmicroRNAs, with each probe being printed in triplicate.

Fourteen negative control probes were designed using the sense sequencesof different microRNAs, chosen from different plants and algae. Twogroups of positive-control probes were used: (i) synthetic small RNAthat were spiked to the RNA before labeling to verify the labelingefficiency, and (ii) probes for abundant small nuclear RNAs (U1, U2, U3,U4, U5, U6, for three plant species and for C. reinhardtii) were spottedon the array to verify the quantity and quality of the RNAs.

Microarray Hybridization and Scanning

Five micrograms of total RNA were labeled by ligation of Cy3 or Cy5 tothe 3′ end. The labeled RNA was mixed with 3× hybridization buffer(Ambion, Austin, Tex.) and hybridized with the slides for 12-16 h in anAgilent Rotational oven at 10-13 rpm. Following hybridization, thearrays were washed twice at room temperature with 1×SSC and 0.2% SDS.Next, the arrays were washed in 0.1×SSC followed by a 1 min wash infresh Agilent Stabilization and Drying solution (Agilent, Santa Clara,Calif.).

Array Signal Calculation and Normalization

Array images were analyzed using the Feature Extraction software (FE)9.5.1 (Agilent, Santa Clara, Calif.). Experiments were repeated fourtimes and triplicate spots were combined to produce one signal for eachprobe by taking the logarithmic mean of reliable spots. All data werelog-transformed (natural base) and the analysis was performed inlog-space. A reference data vector for normalization R was calculated bytaking the median expression level for each probe across nitrogenstarvation samples. For each sample data vector S, a 2nd degreepolynomial F was found, in order to provide the best fit between thesample data and the reference data, such that R≈F(S). For each probe inthe sample (element Si in the vector S), the normalized value (inlog-space) Mi was calculated from the initial value Si by transformingit with the polynomial function F, so that Mi=F(Si). P-values werecalculated using a two-sided t-test on the log-transformed normalizedfluorescence signal. The fold-difference (ratio of the median normalizedfluorescence) was calculated for each microRNA.

Quantitative Real-Time PCR

Differentially expressed microRNAs related to resistance to abioticstress were identified and validated by qPCR. RNA was subjected to apolyadenylation reaction as previously described [Shi R, Chiang V L,Biotechniques (2005) 39:519-525]. Briefly, RNA was incubated in thepresence of a poly A polymerase enzyme (Takara, Otsu Japan), MnCl₂, andATP for 1 h at 37° C. Then, using an oligo dT primer harboring aconsensus sequence, reverse transcription was performed on total RNAusing SuperScript II RT (Invitrogen, Carlsbad, Calif.). Next, the cDNAwas amplified by real-time PCR; this reaction contained amicroRNA-specific forward primer and a universal reverse primercomplementary to the consensus 3′ sequence of the oligo dT tail. Resultsrepresent the median of four repeats with each one done in triplicate,and the signal was normalized against the median of three referencemicroRNAs.

Example 1 Differential Expression of miRNAs in Corn Plants after OneWeek of Drought or High Salinity Conditions

Corn plants were first allowed to grow at standard, optimal conditionsfor two weeks. Plants were subsequently divided into three groups:control, salinity- and drought-treated. The control group was irrigatedto saturation, twice weekly with tap water. The salinity group wasirrigated twice weekly with tap water spiked with 150 mM NaCl. Thedrought group was not irrigated. The experiment continued for one week,after which plants were harvested. Two to three plants from eachtreatment were grouped as a biological repeat. Five to six repeats wereobtained for each treatment, and RNA was extracted from leaf tissue.

The expression level of the corn microRNAs was analyzed by highthroughput microarray to identify microRNAs that were differentiallyexpressed in response to drought or salinity. Several members of themiR-169 family were found to be down-regulated in response to droughtstress. Several members of the miR-167 family were found to beup-regulated and several members of the miR-169 family were found to bedown-regulated in response to salinity stress. The results are presentedin Table 2 below:

TABLE 2 Differentially-expressed microRNAs in corn plants after one weekof treatment Fold miR Hairpin MicroRNA P value Change DirectionTreatment SEQ ID NO SEQ ID NO osa-miR169e 7.7e−003 1.58 Down 150 mM NaCl1 11 ath-miR169a 8.6e−003 1.58 Down 150 mM NaCl 2 12 ptc-miR169o1.0e−002 1.56 Down 150 mM NaCl 3 13 bna-miR169c 1.1e−002 1.53 Down 150mM NaCl 4 14 ptc-miR169v 1.1e−002 1.52 Down 150 mM NaCl 5 15 ath-miR169d1.6e−002 1.53 Down 150 mM NaCl 6 16 ath-miR167c 2.1e−002 1.57 Up 150 mMNaCl 7 17 bna-miR167a 3.0e−002 1.51 Up 150 mM NaCl 8 18 ppt-miR1673.3e−002 1.68 Up 150 mM NaCl 9 19 ptc-miR167f 3.7e−002 1.54 Up 150 mMNaCl 10 20 ppt-miR894 3.4e−003 1.81 Down 150 mM NaCl 21 26 osa-miR164e1.4e−002 1.96 Up 1 week drought 22 27 bna-miR169g 4.2e−003 2.03 Down 1week drought 23 28 ptc-miR169o 1.9e−003 2.00 Down 1 week drought 3 13ptc-miR169v 3.4e−003 1.89 Down 1 week drought 5 15 osa-miR169e 1.4e−0031.72 Down 1 week drought 1 11 ath-miR169d 1.4e−003 1.68 Down 1 weekdrought 6 16 ath-miR169a 4.9e−004 1.68 Down 1 week drought 2 12vvi-miR169m 1.0e−003 1.57 Down 1 week drought 24 29 ptc-miR169q 1.4e−0031.54 Down 1 week drought 25 30

Example 2 Differential Expression of miRNAs in Corn Plants after TwoWeeks of Drought or High Saline Conditions

Corn plants were first allowed to grow at standard, optimal conditionsfor two weeks. Plants were subsequently divided into three groups:control, salinity- and drought-treated. The control group was watered tosaturation twice weekly with tap water. The salinity group was irrigatedtwice weekly with tap water spiked with 150 mM NaCl. The drought groupwas not irrigated. The experiment continued for two weeks, after whichplants were harvested. Two to three plants from each treatment weregrouped as a biological repeat. Five to six repeats were obtained foreach treatment, and RNA was extracted from leaf tissue.

The expression level of the corn microRNAs was analyzed by highthroughput microarray to identify microRNAs that are differentiallyexpressed in response to drought or salinity. Several members of themiR-156 family were found to be up-regulated in response to both saltand drought stress. Several members of the miR-171 family were found tobe down-regulated under drought stress. ath-miR854a was found to bedown-regulated under both stresses. The results are presented in Table 3below:

TABLE 3 Differentially-expressed microRNAs in corn plants after twoweeks of treatment miR Hairpin Fold SEQ SEQ ID MicroRNA P value ChangeDirection Treatment ID NO NO Table 3A: Leaf samples bna-miR156a 1.3e−0021.60 Up 150 mM NaCl 31 49 ath-miR156g 2.6e−002 1.58 Up 150 mM NaCl 32 50osa-miR156l 4.4e−002 1.56 Up 150 mM NaCl 33 51 ath-miR854a 1.5e−002 1.53Down 150 mM NaCl 34 52 ppt-miR1039- 3.7e−004 7.94 Up 2 weeks drought 3553 3p osa-miR156l 5.4e−006 4.31 Up 2 weeks drought 33 51 bna-miR156a2.1e−005 4.14 Up 2 weeks drought 31 49 ath-miR156g 8.7e−007 3.54 Up 2weeks drought 32 50 zma-miR156k 2.2e−005 3.38 Up 2 weeks drought 36 54vvi-miR156e 4.3e−004 3.31 Up 2 weeks drought 37 55 sbi-miR156d 3.9e−0031.87 Up 2 weeks drought 38 56 ath-miR167d 6.2e−003 1.63 Up 2 weeksdrought 39 57 ptc-miR167h 2.3e−002 1.76 Up 2 weeks drought 40 58sbi-miR168 4.7e−004 1.68 Up 2 weeks drought 41 59 gma-miR168 2.6e−0031.56 Up 2 weeks drought 42 60 sof-miR408a 1.6e−002 1.90 Up 2 weeksdrought 43 61 ath-miR408 2.5e−002 1.73 Up 2 weeks drought 44 62ppt-miR160b 1.7e−004 1.50 Down 2 weeks drought 45 63 ppt-miR160c6.7e−004 1.69 Down 2 weeks drought 46 64 osa-miR390 1.6e−002 2.67 Down 2weeks drought 47 65 ppt-miR390c 2.0e−002 2.53 Down 2 weeks drought 48 66ath-miR854a 1.1e−003 1.88 Down 2 weeks drought 34 52 Table 3B: Rootsamples smo-miR171a 7.4e−003 1.61 Down 2 weeks drought 74 68 smo-miR171b1.4e−002 1.62 Down 2 weeks drought 75 69 sbi-miR171b 1.5e−002 1.57 Down2 weeks drought 76 70 zma-miR171c 4.4e−002 1.51 Down 2 weeks drought 7771 zma-miR171f 2.3e−002 1.59 Down 2 weeks drought 78 72 ath-miR854a3.2e−002 1.60 Down 2 weeks drought 34 52 ppt-miR894 1.6e−002 1.55 Down 2weeks drought 21 26

Example 3 Differential Expression of miRNAs in Sorghum Plants after TwoWeeks of Drought or High Saline Conditions

Sorghum bicolor plants were first allowed to grow at standard, optimalconditions for two weeks. Plants were subsequently divided into threegroups: control, salinity- and drought-treated. The control group waswatered to saturation twice a week with tap water. The salinity groupwas irrigated twice weekly with tap water spiked with 150 mM NaCl. Thedrought group was not irrigated. The experiment continued for two weeks,after which plants were harvested. Two to three plants from eachtreatment were grouped as a biological repeat. Five to six repeats wereobtained for each treatment, and RNA was extracted from leaf tissue.

The expression level of the sorghum microRNAs was analyzed by highthroughput microarray to identify microRNAs that were differentiallyexpressed in response to drought or salinity. Several members of themiR-529, miR-164 and miR-159 families were found to be up-regulatedunder drought stress. Several members of the miR-169 families were foundto be down-regulated under drought stress. Several members of themiR-156 family were found to be up-regulated in response to both saltand drought stress, and several members of the miR-171 and miR-172families were found to be down-regulated under both stresses. Theresults are presented in Table 4 below:

TABLE 4 Differentially-expressed microRNAs in sorghum plants after twoweeks of treatment miR Hairpin Fold SEQ ID SEQ ID MicroRNA P valueChange Direction Treatment NO NO Table 4A: Leaf samples smo-miR156c2.8e−003 3.30 Up 150 mM NaCl 79 137 smo-miR156d 9.9e−006 5.23 Up 150 mMNaCl 80 138 zma-miR156k 2.3e−002 2.07 Up 150 mM NaCl 36 54 ppt-miR3953.6e−003 3.43 Up 150 mM NaCl 81 139 ppt-miR1039-3p 3.8e−003 2.73 Up 150mM NaCl 35 53 smo-miR1091 3.6e−003 3.74 Up 150 mM NaCl 82 140tae-miR1118 4.0e−004 5.92 Up 150 mM NaCl 83 141 tae-miR1134 8.3e−0033.29 Up 150 mM NaCl 84 142 sbi-miR171b 1.4e−003 2.38 Down 150 mM NaCl 7670 smo-miR171a 3.6e−003 2.02 Down 150 mM NaCl 74 68 smo-miR171b 1.2e−0032.16 Down 150 mM NaCl 75 69 vvi-miR171a 1.0e−003 2.30 Down 150 mM NaCl85 143 vvi-miR171i 8.8e−004 2.11 Down 150 mM NaCl 86 144 zma-miR171c5.1e−003 2.20 Down 150 mM NaCl 77 71 zma-miR171f 1.3e−003 2.10 Down 150mM NaCl 78 72 gma-miR172a 6.9e−004 2.38 Down 150 mM NaCl 87 145ath-miR172c 4.0e−003 2.55 Down 150 mM NaCl 88 146 zma-miR172e 1.0e−0022.12 Down 150 mM NaCl 89 147 sbi-miR399b 2.2e−002 2.31 Down 150 mM NaCl90 148 osa-miR530-3p 2.1e−003 6.91 Down 150 mM NaCl 91 149 ath-miR854a6.8e−003 2.24 Down 150 mM NaCl 34 52 smo-miR156c 4.4e−003 3.04 Up 2weeks drought 79 137 smo-miR156d 3.7e−004 6.36 Up 2 weeks drought 80 138ppt-miR395 5.5e−003 3.77 Up 2 weeks drought 81 139 ppt-miR529a 5.5e−0032.66 Up 2 weeks drought 92 150 ppt-miR529d 3.2e−003 2.25 Up 2 weeksdrought 93 151 ppt-miR529e 1.8e−003 2.53 Up 2 weeks drought 94 152ppt-miR529g 1.0e−002 2.18 Up 2 weeks drought 95 153 ppt-miR1039-3p1.5e−004 5.49 Up 2 weeks drought 35 53 smo-miR1091 1.3e−003 5.81 Up 2weeks drought 82 140 tae-miR1118 1.7e−003 7.64 Up 2 weeks drought 83 141tae-miR1134 8.4e−003 4.24 Up 2 weeks drought 84 142 osa-miR169a 2.6e−0022.01 Down 2 weeks drought 96 154 sbi-miR169b 1.8e−002 2.00 Down 2 weeksdrought 97 155 bna-miR169m 1.1e−002 2.37 Down 2 weeks drought 98 156vvi-miR171a 7.4e−003 2.52 Down 2 weeks drought 85 143 sbi-miR171b5.6e−004 2.43 Down 2 weeks drought 76 70 zma-miR171c 3.4e−003 2.28 Down2 weeks drought 77 71 gma-miR172a 1.8e−002 2.22 Down 2 weeks drought 87145 ath-miR172c 2.6e−002 2.27 Down 2 weeks drought 88 146 zma-miR172e3.5e−002 2.06 Down 2 weeks drought 89 147 ath-miR395a 4.4e−002 2.56 Down2 weeks drought 99 157 bna-miR397a 1.2e−002 2.97 Down 2 weeks drought100 158 ptc-miR397b 9.1e−003 3.28 Down 2 weeks drought 101 159smo-miR408 1.3e−003 2.57 Down 2 weeks drought 102 160 osa-miR5282.5e−003 2.71 Down 2 weeks drought 103 161 osa-miR530-3p 9.9e−006 14.00Down 2 weeks drought 91 149 Table 4B: Root samples smo-miR171a 2.4e−0032.26 Down 150 mM NaCl 74 68 vvi-miR171a 7.1e−004 2.18 Down 150 mM NaCl85 143 ath-miR171b 3.7e−004 2.39 Down 150 mM NaCl 104 162 sbi-miR171b1.1e−003 2.09 Down 150 mM NaCl 76 70 smo-miR171b 2.4e−003 2.43 Down 150mM NaCl 75 69 zma-miR171f 2.7e−003 2.22 Down 150 mM NaCl 78 72ppt-miR896 5.6e−003 2.17 Down 150 mM NaCl 105 163 smo-miR156d 2.8e−0022.02 Up 2 weeks drought 80 138 pta-miR159c 7.0e−005 7.12 Up 2 weeksdrought 106 164 sof-miR159c 1.7e−003 2.84 Up 2 weeks drought 107 165osa-miR159c 2.3e−003 2.29 Up 2 weeks drought 108 166 osa-miR159d4.0e−003 2.20 Up 2 weeks drought 109 167 osa-miR164a 5.4e−004 2.41 Up 2weeks drought 110 168 sbi-miR164b 2.9e−004 2.47 Up 2 weeks drought 111169 osa-miR164c 1.7e−004 2.48 Up 2 weeks drought 112 170 ptc-miR164f1.4e−004 2.60 Up 2 weeks drought 113 171 ppt-miR1039-3p 1.9e−003 5.10 Up2 weeks drought 35 53 tae-miR1129 1.1e−002 2.16 Up 2 weeks drought 114172 sbi-miR169c 2.9e−002 2.11 Down 2 weeks drought 117 175 sbi-miR169i2.9e−003 4.19 Down 2 weeks drought 118 176 ptc-miR169x 3.7e−002 2.22Down 2 weeks drought 119 177 smo-miR171a 6.7e−003 2.06 Down 2 weeksdrought 74 68 smo-miR171b 5.9e−003 2.16 Down 2 weeks drought 75 69smo-miR408 7.6e−003 2.16 Down 2 weeks drought 102 160

Example 4 Differential Expression of miRNAs in Corn Plants after SixDays of Drought or High Saline Conditions

Corn plants were first allowed to grow at standard, optimal conditionsfor two weeks. Plants were subsequently divided into three groups:control, salinity- and drought-treated. The control group was watered tosaturation twice weekly with tap water. The salinity group was irrigatedtwice weekly with tap water spiked with 300 mM NaCl. The drought groupwas not irrigated. The experiment continued for six days after, whichplants were harvested. Two to three plants from each treatment weregrouped as a biological repeat. Five to six repeats were obtained foreach treatment, and RNA was extracted from leaf tissue.

The expression level of the corn microRNAs was analyzed by highthroughput microarray to identify microRNAs that were differentiallyexpressed in response to drought or salinity. Several members of themiR-156 family were found to be up-regulated under drought stress.Several members of the miR-167 family were found to be up-regulatedunder salt stress. Several members of the miR-164 family were found tobe up-regulated in response to both salt and drought stress, and severalmembers of the miR-399 family were found to be down-regulated under bothstresses. The results are presented in Table 5 below:

TABLE 5 Differentially-expressed microRNAs in corn plants after six daysof treatment miR Hairpin Fold SEQ ID SEQ ID MicroRNA P value ChangeDirection Treatment NO NO Table 5A: Leaf samples ath-miR156g 4.5e−0022.61 Up 300 mM NaCl 32 50 ptc-miR164f 4.4e−002 1.60 Up 300 mM NaCl 113171 ath-miR167c 2.6e−003 1.76 Up 300 mM NaCl 7 17 ath-miR167d 5.8e−0031.64 Up 300 mM NaCl 39 57 ptc-miR167f 9.6e−003 1.59 Up 300 mM NaCl 10 20ptc-miR167h 1.2e−002 1.67 Up 300 mM NaCl 40 58 ppt-miR1039-3p 2.6e−0062.58 Up 300 mM NaCl 35 53 sbi-miR171e 2.7e−005 5.46 Down 300 mM NaCl 120178 sbi-miR171f 1.5e−006 5.42 Down 300 mM NaCl 121 179 osa-miR3901.1e−003 1.91 Down 300 mM NaCl 47 65 sbi-miR399b 1.3e−004 3.47 Down 300mM NaCl 90 148 mtr-miR399d 1.2e−004 3.06 Down 300 mM NaCl 122 180smo-miR408 4.4e−003 1.92 Down 300 mM NaCl 102 160 ppt-miR477a-3p1.2e−002 1.71 Down 300 mM NaCl 123 181 osa-miR528 1.9e−004 4.31 Down 300mM NaCl 103 161 ath-miR855 2.5e−003 2.54 Down 300 mM NaCl 124 182ppt-miR1026a 9.2e−003 1.98 Down 300 mM NaCl 125 183 bna-miR156a 1.7e−0032.62 Up 6 days drought 31 49 ath-miR156g 1.8e−003 2.44 Up 6 days drought32 50 osa-miR156l 2.1e−003 2.68 Up 6 days drought 33 51 zma-miR156k2.4e−003 2.33 Up 6 days drought 36 54 osa-miR164a 3.0e−003 1.90 Up 6days drought 110 168 sbi-miR164b 1.6e−003 1.97 Up 6 days drought 111 169ptc-miR164f 2.6e−003 1.97 Up 6 days drought 113 171 ppt-miR1039-3p4.6e−004 1.61 Up 6 days drought 35 53 sbi-miR171f 4.7e−004 1.78 Down 6days drought 121 179 sbi-miR399b 1.2e−002 1.98 Down 6 days drought 90148 osa-miR528 1.4e−003 2.34 Down 6 days drought 103 161 ath-miR8551.8e−003 2.08 Down 6 days drought 124 182 ppt-miR901 2.6e−002 2.04 Down6 days drought 126 184 Table 5B: Root samples osa-miR164a 9.6e−004 1.67Up 300 mM NaCl 110 168 sbi-miR164b 1.7e−003 1.63 Up 300 mM NaCl 111 169osa-miR164c 3.0e−003 1.62 Up 300 mM NaCl 112 170 tae-miR1129 4.1e−0031.57 Up 300 mM NaCl 114 172 ppt-miR1039-3p 4.7e−003 2.87 Up 300 mM NaCl35 53 sbi-miR166e 5.1e−003 1.93 Down 300 mM NaCl 127 185 osa-miR3903.8e−005 1.86 Down 300 mM NaCl 47 65 sbi-miR399a 1.5e−004 2.38 Down 300mM NaCl 128 186 mtr-miR399d 3.5e−004 2.67 Down 300 mM NaCl 122 180sbi-miR399b 1.2e−004 2.61 Down 300 mM NaCl 90 148 ptc-miR156k 1.8e−0041.58 Up 6 days drought 129 187 osa-miR164a 2.7e−004 1.52 Up 6 daysdrought 110 168 sbi-miR164b 1.5e−004 1.52 Up 6 days drought 111 169tae-miR1129 1.3e−005 1.88 Up 6 days drought 114 172 sbi-miR166e 4.6e−0031.53 Down 6 days drought 127 185 sbi-miR169c 5.5e−003 1.57 Down 6 daysdrought 117 175 sbi-miR399a 1.3e−002 2.19 Down 6 days drought 128 186sbi-miR399b 1.1e−002 2.19 Down 6 days drought 90 148 mtr-miR399d1.6e−002 2.33 Down 6 days drought 122 180 vvi-miR535a 1.6e−002 1.89 Down6 days drought 131 189

Example 5 Method for Generating Transgenic Plants with ManipulatedExpression of Arabidopsis microRNAs

Synthetic DNA fragments were synthesized by Genscript® (GenScript USAInc., Piscataway, N.J., USA) and cloned into the pUC57 vector. Theseclones contained, respectively, the hairpins of Arabidopsis microRNAs,flanked by 100-300 nucleotides of genomic DNA, as follows: ath-miR167a(SEQ ID NO: 193), ath-miR156a (SEQ ID NO: 191), ath-miR164a (SEQ ID NO:192), and ath-miR169a (SEQ ID NO: 12). The DNA fragments were designedto contain BamHI and KpnI restriction enzyme recognition sites at the 5′and 3′ ends, respectively. SEQ ID NOS: 191-193 contain within theirhairpin sequence miRNAs which are identical to bna-miR167a (SEQ ID NO:8), bna-miR156a (SEQ ID NO: 31) and osa-miR164a (SEQ ID NO: 110),respectively.

Each fragment was digested with BamHI and KpnI and ligated into the pOREE2 binary vector (Accession number: AY562535) previously digested withthe same enzymes to drive the expression of the microRNA hairpins underthe regulation of the hydroperoxide lyase (HPL) promoter. The HPLpromoter is known to drive constitutive high-level expression oftransgenes in dicot plants. It is known to be active in Arabidopsis,tobacco, cauliflower, soybean, alfalfa, peach, white spruce, wheat andgrape. The resulting vectors were designated pORE156, pORE164, pORE167and pORE169.

Each of the vectors was transformed by electroporation intoAgrobacterium tumefaciens strain GV3101. Single colonies were grown andused to transform Arabidopsis thaliana plants, ecotype Colombia, usingthe floral dip method (Clough and Bent, The Plant Journal (1998) 16(6)735-743). In this method, Agrobacterium was grown in appropriate growthmedium and suspended in infiltration medium, into which plants weresubsequently immersed. Seeds from the plants were collected andsurface-sterilized, then plated on Petri dishes in Arabidopsis seedmedium plus kanamycin and agarose. Transgenic plants became visible andwere distinguished after about two weeks.

Transgenic plants are analyzed by PCR to validate integration of thetransgene into the genome and by quantitative RT-PCR to validateover-expression of the relevant mature microRNA. Salinity and droughttolerance of the transgenic plants is compared to that of the wild type.Transgenic plants with relatively enhanced resistance to abiotic stressare identified.

Example 6 Method for Generating Transgenic Plants with Reduced microRNARegulation

Target prediction enables manipulation of microRNA regulation byintroducing silent mutations into the microRNA-binding site, leading tothe expression of a microRNA-resistant target, thereby bypassingmicroRNA regulation. Alternatively, manipulation of microRNA regulationcan be performed by microRNA over-expression. Both these strategies havebeen used in plants and have resulted in significant phenotypealterations.

Reducing microRNA regulation of target genes can potentially be achievedby two methods, either by expressing a microRNA-resistant gene or byexpressing a target-mimic sequence.

Expressing a microRNA-Resistant Target

In this method, silent mutations are introduced in the microRNA bindingsite of the target gene so that the DNA and resulting RNA sequences arechanged in a way that prevents microRNA binding, but the amino acidsequence of the protein is unchanged. For example, Arabidopsis miR-169a(5′ CAGCCAAGGAUGACUUGCCGA 3′, SEQ ID NO: 2) is predicted to targetseveral CCAAT-binding transcription factors. The predicted binding sitefor one of the targets (gene ID: 838335 NF-YA8) is as follows: 5′ACGGGAAGTCATCCTTGGCTA 3′ (SEQ ID NO: 497).

A new sequence can be synthesized instead of the existing binding site,in which the DNA sequence is changed, but the translated amino acidsequence is retained, for example: 5′ ATGGTAAAAGCAGTCTAGAGC 3′ (SEQ IDNO: 498); the DNA sequence of the rest of the gene is left unchanged.Hybridization of the new sequence with miR-169a is impossible.

This new gene can be introduced into Arabidopsis plants and will expressa functional gene, which is immune to miR-169a regulation and encodes afunctional NF-YA8 protein.

Expressing a Target-Mimic Sequence

Plant microRNAs usually lead to cleavage of their targeted gene, withthis cleavage typically occurring between bases 10 and 11 of themicroRNA. This position is therefore especially sensitive to mismatchesbetween the microRNA and the target. It was found that expressing a DNAsequence that could potentially be targeted by a microRNA, but containstwo extra nucleotides between the two nucleotides that are predicted tohybridize with bases 10-11 of the microRNA (thus creating a bulge inthat position), can inhibit the regulation of that microRNA on itsnative targets, as shown in FIG. 1.

This type of sequence is referred to as a “target-mimic”. Inhibition ofthe microRNA regulation is presumed to occur through physicallycapturing the microRNA by the target-mimic sequence and titering-out themicroRNA, thereby reducing its abundance. This method was previouslyused to reduce the amount and, consequentially, the regulation ofmicroRNA 399 in Arabidopsis [Franco-Zorilla J M et al., Nature Genetics(2007) 39(8):1033-1037].

Predicted targets for regulating miRNA expression by sequence alterationin the transgenic plants of the present invention are shown for corn(Table 6) and sorghum (Table 7).

TABLE 6 Predicted miR targets in corn (Zea mays) Mir Predicted target inFamily Sorghum SEQ ID NO: ncbi accession #: protein id SEQ ID NO:zma-169 TC386981 195 BT061088.1 ACN25785 342 TC398825 196 NM_001176546.1NP_001170017 343 TC391807 197 NM_001155603.1 NP_001149075 344 TC396785198 BT038709.1 ACF83714 345 TC398710 199 EU961865.1 TC374958 200BT066403.1 ACN33300 346 TC414302 201 BT036648.1 ACF81653 347 TC379185202 BT054250.1 ACL52857 348 TC402489 203 BT062100.1 ACN26797 349TC409629 204 NM_001139400.1 NP_001132872 350 zma-167 CF630597 205GQ905541.1 TC384517 206 HM004518.1 ADG43137 351 TC390155 207XM_002447260.1 XP_002447305 352 TC398618 208 NM_001176880.1 NP_001170351353 TC410716 209 HM004518.1 ADG43137 354 CO439534 210 NM_001175558.1NP_001169029 355 TC400356 211 AY110452.1 TC402680 212 AY108832.1TC414084 213 NM_001196958.1 NP_001183887 356 TC433424 214 AY110452.1ppt- CF630597 215 miR894 TC384517 216 XM_002447260.1 XP_002447305 357TC390155 217 XM_002447260.1 XP_002447305 358 TC398618 218 NM_001176880.1NP_001170351 359 TC410716 219 HM004518.1 ADG43137 360 CO439534 220NM_001175558.1 NP_001169029 361 TC400356 221 AY110452.1 TC402680 222AY108832.1 TC414084 223 NM_001196958.1 NP_001183887 362 TC433424 224AY110452.1 zma-164 TC372777 225 AJ833967.1 CAH56058 363 TC372793 226AJ833966.1 CAH56057 364 TC393990 227 NM_001175536.1 NP_001169007 365TC375804 228 NM_001196213.1 NP_001183142 366 TC402142 229 NM_001147702.1NP_001141174 367 CO452388 230 EU971666.1 ACG43784 368 TC385354 231NM_001136787.1 NP_001130259 369 TC422132 232 NM_001175843.1 NP_001169314370 TC403112 233 NM_001174985.1 NP_001168456 371 DY689161 234NM_001153167.1 NP_001146639 372 TC384685 235 BT065898.1 ACN31774 373zma-156 TC374118 236 BT041777.2 ACF86782 374 TC375695 237 EU965000.1ACG37118 375 TC378352 238 XM_002456814.1 XP_002456859 376 TC383848 239EU965000.1 ACG37118 377 TC384479 240 BT054654.1 ACL53261 378 TC386709241 AJ011618.1 CAB56631 379 TC387392 242 NM_001152255.1 NP_001145727 380TC391022 243 BT084089.1 ACR34442 381 TC401092 244 NM_001143577.1NP_001137049 382 TC388259 282 BT069447.1 ACN36344 417 smo-1091 TC373124283 BT064902.1 ACN30778 418 TC420039 284 BT086539.1 ACR36892 419zma-399d TC372604 285 NM_001112347.1 NP_001105817 420 TC384393 286BT086308.1 ACR36661 421 TC405480 287 NM_001112347.1 NP_001105817 422EE168670 288 NM_001112347.1 NP_001105817 423 osa-530- TC394057 289BT054711.1 ACL53318 424 3p TC399070 290 AY111547.1 EE681052 291NM_001155648.1 NP_001149120 425 TC423023 292 BT041995.2 ACF87000 426TC423251 293 EU955378.1 ACG27496 427 CB833661 294 BT041995.2 ACF87000428 DR786428 295 BT066466.1 ACN33363 429 ppt-477a- CF048906 296NM_001148796 NP_001142268 430 3p TC380158 297 BT038098 ACF83103 431TC373894 298 BT054486 ACL53093 432 TC417766 299 BT054486 ACL53093 433EE042910 300 BT040656 ACF85661 434 TC382335 301 NM_001147977NP_001141449 435 zma-395 TC391887 302 BT063912.1 ACN28609 436 TC391300303 NM_001111407.1 NP_001104877 437 CO462284 304 BT067126.1 ACN34023 438ath855 TC373849 305 NM_001157122.1 NP_001150594 439 TC375345 306BT035131.1 ACF80136 440 CO445522 307 BT035131.1 ACF80136 441 ppt-1039-BM339650 308 EU943793.1 3p zma-168 TC374776 309 NM_001176734.1NP_001170205 442 TC413096 310 XM_002440366.1 XP_002440411 443 CO459887311 XM_002440366.1 XP_002440411 444 EE285427 312 NM_001175810.1NP_001169281 445 ppt-529g TC372784 313 AY883559.2 AAX83872 446 TC429571314 AY883560.1 AAX83873 447 TC432052 315 AY883559.2 AAX83872 448TC401092 316 NM_001143577.1 NP_001137049 449 TC378352 317 XM_002456814.1XP_002456859 450 TC441933 318 BT064694.1 ACN30570 451 TC397772 319NM_001152261.1 NP_001145733 452 TC374506 320 NM_001139359.2 NP_001132831453 osa-528 TC378699 321 NM_001152326 NP_001145798 454 TC372588 322NM_001112445 NP_001105915 455 TC372613 323 NM_001112451 NP_001105921 456ppt-896 TC406311 324 EU959481.1 ACG31599 457 zma-159 BM338067 325ACG30664.1 ACG30664 458 TC383580 326 NM_001137160.1 NP_001130632 459TC378278 327 NM_001148578.1 NP_001142050 460 TC375562 328 NM_001148578NP_001142050 461 TC398538 329 NM_001148578.1 NP_001142050 462 TC429458330 NM_001148578.1 NP_001142050 463 CO439496 331 NM_001148578.1NP_001142050 464 tae-1129 TC377131 332 NM_001148336 NP_001141808 465ppt-1026a XM_001761787.1 333 XM_001761787.1 XP_001761839 466 ppt-901TC404127 334 AY108169 zma-166 TC372571 335 BT066287.1 ACN32163 467TC374219 336 NM_001112524.1 NP_001105994 468 TC383262 337 NM_001148922.1NP_001142394 469 DV510458 338 NM_001152743.1 NP_001146215 470 EE185687339 NM_001152743.1 NP_001146215 471 osa-535 TC395045 340 NM_001148293.1NP_001141765 472 DV028665 341 BT065661.1 ACN31537 473

TABLE 7 Predicted miR targets in sorghum Mir Predicted target SEQ ID SEQID Family in Sorghum NO: ncbi accession #: protein id NO: ppt-395TC115368 474 XM_002448709 XP_002448754 486 TC128625 475 EU962499 5279979476 XM_002461779 XP_002461824 487 abi-397 TC129073 477 BT064158 ACN28855488 CF429403 478 XM_002458701.1 XP_002458746 489 5283185 479XM_002458702.1 XP_002458747 490 5279496 480 XM_002465904.1 XP_002465949491 5289814 481 XM_002439871.1 XP_002439916 492 5259332 482XM_002465440.1 XP_002465485 493 smo-1091 TC121467 483 XM_002465662.1XP_002465707 494 tae-1134 5279991 484 XM_002459659.1 XP_002459704 495CF488307 485 XM_002459659.1 XP_002459704 496

Example 7 miR-156a and miR-169a Transgenic Plants Comprising EnhancedDrought Tolerance

Native hairpins of miRs ath-156a and ath-169a were synthesized byGenscript (order no. 72356) with BamHI and KpnI restriction sites at thebeginning and the end of the gene, respectively. The 312 bp fragmentswere excised and inserted into pORE-E2 plasmid using BamHI and KpnIrestriction enzymes in sequential digest (as described in Example 5,above). RBC (Real Biotech Corporation) E. coli DH5a competent cells weretransformed with these ligations. Colony PCR on kanamycin resistantcolonies was performed using miR specific primer and M13rev universalprimer (present in pORE-E2). pORE-E2-156a colony #4 and pORE-E2-169acolony #1 were chosen for further work after plasmid purification usingkit and sequencing of the plasmid to verify correct sequence.

Home-made competent Agrobacterium tumefaciens were transformed with theplasmids. Bacteria were shaken in 28° C. for 2 hours for regeneration,and then plated on LB+kanamycin 50 μg/ml for selection of resistantcolonies for 48 hours. Colonies were then subjected to PCR forverification of plasmid integration using miR specific rev primer andpORE-E2 specific primer. Colony #1 for each plasmid was chosen forplants transformation.

Arabidopsis thaliana seeds were sown in pots, and then incubated in 4°C. for 2 days. Trays were then moved into growth rooms (20-22° C., longday—16 hours light, 8 hours dark). After 3 weeks plants were cut toenable growth of many branches. After a week, the plants were taken fortransformation. Agrobacteria carrying the plasmids were grown for 3 daysprior to transformation. On transformation day, bacteria were re-checkedfor correct plasmid presence by PCR. Agrobacteria were precipitated andthen re-suspended in infiltration medium (10 mM MgCl2, 5% Sucrose, 0.044μM BAP and 0.03% Tween 20) in deep bowls. The well watered plants wereflipped over into the liquid until all flowers were sunken. After 3minutes, pots were taken out and were laid on both sides on filteredpaper to absorb liquids. After 24 hours, pots were straightened andwatered. Plants were left to develop seeds in the growth rooms. Seedswere collected after a few weeks, and then were germinated on GM agarplates containing kanamycin 50 μg/ml for selection of resistant plants.Resistant seedling were transferred to pots and grown for two moregeneration. Homozygous plants of each plasmid were chosen for furthercharacterization of expression of miR156a and miR169a (156-1-1 and169-3-28, respectively).

As is illustrated in FIGS. 2A-B, 3A-B, 4, 5A-C and 6A-B 156-1-1,169-3-28 transgenic plants and wild-type plants were grown for 3 weeksunder the same conditions (as described above). Plants of each line werethen divided into two separate trays; one tray was watered normally andthe other was deprived of water for 10 days. After 10 days, thedehydrated trays were watered again in two day intervals.

As shown in FIGS. 2A and 3A, control watered trays of all three linesdisplayed similar characteristics: the wild-type and the transgenicplants all have similar sizes, number of branches and flowers. However,as is illustrated in FIGS. 2B, 3B, 4, 5A-C and 6A-B, the wild-typeplants could not recover from the de-hydration, and all the wild-typeplants died, in sharp contrast, most of the transgenic plants recoveredand displayed characteristics similar to the hydrated control plants.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by into thespecification, to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting.

What is claimed is:
 1. A method of increasing tolerance of a plant to anabiotic stress or increasing biomass, vigor or yield of a plant, themethod comprising upregulating within the plant an exogenouspolynucleotide of a microRNA or a precursor thereof, wherein saidmicroRNA is selected from the group consisting of miR-156, miR-169,miR-164, miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a,ath-miR408, miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129, therebyincreasing the tolerance of the plant to the abiotic stress orincreasing the biomass, vigor or yield of the plant.
 2. The method ofclaim 1, wherein said upregulating is effected by expressing within theplant said exogenous polynucleotide of said microRNA or said precursorthereof.
 3. A method of increasing tolerance of a plant to an abioticstress or increasing biomass, vigor or yield of a plant, the methodcomprising expressing within the plant an exogenous polynucleotideencoding a nucleic acid agent capable of downregulating expression of atarget gene of a microRNA or a precursor thereof, wherein said microRNAis selected from the group consisting of miR-156, miR-169, miR-164,miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408,miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129, thereby increasingthe tolerance of the plant to the abiotic stress or increasing thebiomass, vigor or yield of the plant.
 4. A method of increasingtolerance of a plant to an abiotic stress or increasing biomass, vigoror yield of a plant, the method comprising expressing within the plantan exogenous polynucleotide encoding a nucleic acid agent capable ofdownregulating expression or activity of a microRNA or a precursorthereof, wherein said microRNA is selected from the group consisting ofmiR-171, miR-172, miR-399, miR-854, miR-894, miR-160, miR-166, miR-390,ath-miR395a, smo-miR408, miR-397, miR-477, miR-528, miR-530, miR-535,miR-855, miR-894, miR-896, miR-901 and miR-1026, thereby increasing thetolerance of the plant to the abiotic stress or increasing the biomass,vigor or yield of the plant.
 5. A method of increasing tolerance of aplant to an abiotic stress or increasing biomass, vigor or yield of aplant, the method comprising expressing within the plant an exogenouspolynucleotide for upregulating expression of a target gene of amicroRNA or a precursor thereof, wherein said microRNA is selected fromthe group consisting of miR-171, miR-172, miR-399, miR-854, miR-894,miR-160, miR-166, miR-390, ath-miR395a, smo-miR408, miR-397, miR-477,miR-528, miR-530, miR-535, miR-855, miR-894, miR-896, miR-901 andmiR-1026, thereby increasing the tolerance of the plant to the abioticstress or increasing the biomass, vigor or yield of the plant.
 6. Themethod of any one of claims 1-5, further comprising growing the plantunder abiotic stress conditions.
 7. The method of any one of claims 1-6,wherein said abiotic stress is selected from the group consisting ofsalinity, water deprivation, low temperature, high temperature, heavymetal toxicity, anaerobiosis, nutrient deficiency, nutrient excess,atmospheric pollution and UV irradiation.
 8. The method of any one ofclaims 2-5, wherein said expressing is effected by transforming a cellof said plant with said exogenous polynucleotide.
 9. The method of claim8, wherein said transforming is effected by introducing into said cellof said plant a nucleic acid construct including said exogenouspolynucleotide and at least one promoter capable of directingtranscription of said exogenous polynucleotide in said cell of saidplant.
 10. The method of any one of claims 2-5, wherein said expressingis effected by infecting said plant with a bacteria comprising saidexogenous polynucleotide.
 11. The method of claim 4, wherein saiddownregulating activity of said microRNA is effected by introducing intosaid plant a target mimic or a micro-RNA resistant target which is notcleaved by said microRNA.
 12. The method of claim 11, wherein saidtarget mimic or said micro-RNA resistant target is essentiallycomplementary to said microRNA provided that one or more of followingmismatches are allowed: (a) a mismatch between the nucleotide at the 5′end of said microRNA and the corresponding nucleotide sequence in saidtarget mimic or said micro-RNA resistant target; (b) a mismatch betweenany one of the nucleotides in position 1 to position 9 of said microRNAand the corresponding nucleotide sequence in said target mimic or saidmicro-RNA resistant target; or (c) three mismatches between any one ofthe nucleotides in position 12 to position 21 of said microRNA and thecorresponding nucleotide sequence in said target mimic or said micro-RNAresistant target provided that there are no more than two consecutivemismatches.
 13. The method of claim 11, wherein said target mimic orsaid micro-RNA resistant target is introduced into a cell of said plantin a nucleic acid construct including a target gene and at least onepromoter capable of directing transcription of said targetpolynucleotide in said cell of said plant.
 14. The method of claim 3, 5or 13, wherein said target gene of said microRNA is as set forth in SEQID NOs: 195-341, 474-485.
 15. A nucleic acid construct, comprising apolynucleotide at least 90% homologous to a nucleic acid sequenceselected from the group consisting of miR-156, miR-169, miR-164,miR-159, miR-167, miR-529, miR-168, ppt-miR395, sof-miR408a, ath-miR408,miR-1039, miR-1091, miR-1118, miR-1134 and miR-1129 or a precursorthereof, wherein said nucleic acid sequence is under a transcriptionalcontrol of at least one promoter capable of directing transcription ofthe polynucleotide in a host cell.
 16. A nucleic acid construct,comprising a polynucleotide at least 90% homologous to a nucleic acidsequence selected from the group consisting of a target gene of miR-171,a target gene of miR-172, a target gene of miR-399, a target gene ofmiR-854, a target gene of miR-894, a target gene of miR-160, a targetgene of miR-166, a target gene of miR-390, a target gene of ath-miR395a,a target gene of smo-miR408, a target gene of miR-397, a target gene ofmiR-477, a target gene of miR-528, a target gene of miR-530, a targetgene of miR-535, a target gene of miR-855, a target gene of miR-894, atarget gene of miR-896, a target gene of miR-901 and a target gene ofmiR-1026, wherein said nucleic acid sequence is under a transcriptionalcontrol of at least one promoter capable of directing transcription ofthe polynucleotide in a host cell.
 17. A nucleic acid construct,comprising a nucleic acid sequence for down-regulating an expression ofa target gene of a microRNA or a precursor thereof, wherein said targetgene of said microRNA is selected from the group consisting of a targetgene of miR-156, a target gene of miR-169, a target gene of miR-164, atarget gene of miR-159, a target gene of miR-167, a target gene ofmiR-529, a target gene of miR-168, a target gene of ppt-miR395, a targetgene of sof-miR408a, a target gene of ath-miR408, a target gene ofmiR-1039, a target gene of miR-1091, a target gene of miR-1118, a targetgene of miR-1134 and a target gene of miR-1129, wherein said nucleicacid sequence is under a transcriptional control of at least onepromoter capable of directing transcription of the polynucleotide in ahost cell.
 18. A nucleic acid construct, comprising a nucleic acidsequence for down-regulating an expression of a microRNA or a precursorthereof, wherein said microRNA is selected from the group consisting ofmiR-171, miR-172, miR-399, miR-854, miR-894, miR-160, miR-166, miR-390,ath-miR395a, smo-miR408, miR-397, miR-477, miR-528, miR-530, miR-535,miR-855, miR-894, miR-896, miR-901 and miR-1026, wherein said nucleicacid sequence is under a transcriptional control of at least onepromoter capable of directing transcription of the polynucleotide in ahost cell.
 19. The nucleic acid construct of any of claims 15-18,wherein said host cell comprises a plant cell.
 20. The nucleic acidconstruct of claim 17, wherein said target gene of miR-169 comprises aNF-YA8 protein.
 21. The method of claim 1 or 3 or nucleic acid constructof claim 15, wherein said miR-156 is selected from the group consistingof bna-miR156a, smo-miR156c, sbi-miR156d, smo-miR156d, vvi-miR156e,ath-miR156g, ptc-miR156k, zma-miR156k and osa-miR156l.
 22. The method ofclaim 1 or 3 or nucleic acid construct of claim 15, wherein said miR-169is selected from the group consisting of ath-miR169a, osa-miR169a,sbi-miR169b, bna-miR169c, sbi-miR169c, ath-miR169d, osa-miR169e,bna-miR169g, sbi-miR169i, bna-miR169m, vvi-miR169m, ptc-miR169o,ptc-miR169q, ptc-miR169v and ptc-miR169x.
 23. The method of claim 1 or 3or nucleic acid construct of claim 15, wherein said miR-164 is selectedfrom the group consisting of osa-miR164a, sbi-miR164b, osa-miR164c,osa-miR164e and ptc-miR164f.
 24. The method of claim 1 or 3 or nucleicacid construct of claim 15, wherein said miR-167 is selected from thegroup consisting of ppt-miR167, bna-miR167a, ath-miR167c, ath-miR167d,ptc-miR167f and ptc-miR167h.
 25. The method of claim 1 or 3 or nucleicacid construct of claim 15, wherein said miR-1039 comprisesppt-miR1039-3p.
 26. The method of claim 1 or 3 or nucleic acid constructof claim 15, wherein said miR-168 is selected from the group consistingof sbi-miR168 and gma-miR168.
 27. The method of claim 1 or 3 or nucleicacid construct of claim 15, wherein said miR-159 is selected from thegroup consisting of pta-miR159c, sof-miR159c, osa-miR159c andosa-miR159d.
 28. The method of claim 1 or 3 or nucleic acid construct ofclaim 15, wherein said miR-529 is selected from the group consisting ofppt-miR529a, ppt-miR529d, ppt-miR529e and ppt-miR529g.
 29. The method ofclaim 1 or 3 or nucleic acid construct of claim 15, wherein saidmiR-1118 comprises tae-miR1118.
 30. The method of claim 1 or 3 ornucleic acid construct of claim 15, wherein said miR-1134 comprisestae-miR1134.
 31. The method of claim 1 or 3 or nucleic acid construct ofclaim 15, wherein said miR-1129 comprises tae-miR1129.
 32. The method ofclaim 1 or 3 or nucleic acid construct of claim 15, wherein saidmiR-1091 comprises smo-miR1091.
 33. The method of claim 4 or 5 ornucleic acid construct of claim 18, wherein said miR-171 is selectedfrom the group consisting of smo-miR171a, vvi-miR171a, ath-miR171b,sbi-miR171b, smo-miR171b, zma-miR171c, sbi-miR171e, sbi-miR171f,zma-miR171f and vvi-miR171i.
 34. The method of claim 4 or 5 or nucleicacid construct of claim 18, wherein said miR-172 is selected from thegroup consisting of gma-miR172a, ath-miR172c and zma-miR172e.
 35. Themethod of claim 4 or 5 or nucleic acid construct of claim 18, whereinsaid miR-854 comprises ath-miR854a.
 36. The method of claim 4 or 5 ornucleic acid construct of claim 18, wherein said miR-894 comprisesppt-miR894.
 37. The method of claim 4 or 5 or nucleic acid construct ofclaim 18, wherein said miR-160 is selected from the group consisting ofppt-miR160b and ppt-miR160c.
 38. The method of claim 4 or 5 or nucleicacid construct of claim 18, wherein said miR-390 is selected from thegroup consisting of osa-miR390 and ppt-miR390c.
 39. The method of claim4 or 5 or nucleic acid construct of claim 18, wherein said miR-399 isselected from the group consisting of sbi-miR399a, sbi-miR399b andmtr-miR399d.
 40. The method of claim 4 or 5 or nucleic acid construct ofclaim 18, wherein said miR-166 comprises sbi-miR166e.
 41. The method ofclaim 4 or 5 or nucleic acid construct of claim 18, wherein said miR-397is selected from the group consisting of bna-miR397a and ptc-miR397b.42. The method of claim 4 or 5 or nucleic acid construct of claim 18,wherein said miR-477 comprises ppt-miR477a-3p.
 43. The method of claim 4or 5 or nucleic acid construct of claim 18, wherein said miR-528comprises osa-miR528.
 44. The method of claim 4 or 5 or nucleic acidconstruct of claim 18, wherein said miR-530 comprises osa-miR530-3p. 45.The method of claim 4 or 5 or nucleic acid construct of claim 18,wherein said miR-535 comprises vvi-miR535a.
 46. The method of claim 4 or5 or nucleic acid construct of claim 18, wherein said miR-855 comprisesath-miR855.
 47. The method of claim 4 or 5 or nucleic acid construct ofclaim 18, wherein said miR-896 comprises ppt-miR896.
 48. The method ofclaim 4 or 5 or nucleic acid construct of claim 18, wherein said miR-901comprises ppt-miR901.
 49. The method of claim 4 or 5 or nucleic acidconstruct of claim 18, wherein said miR-1026 comprises ppt-miR1026a. 50.A plant cell comprising the nucleic acid nucleic acid construct of anyof claims 15-18.
 51. A plant or a portion thereof comprising the nucleicacid construct of any of claims 15-18.
 52. A food or feed comprising theplant of claim 51 or a portion thereof.
 53. A method of evaluating atrait of a plant, the method comprising: (a) expressing in a plant or aportion thereof the nucleic acid construct of any of claims 15-18; and(b) evaluating a trait of a plant as compared to a wild type plant ofthe same type; thereby evaluating the trait of the plant.
 54. The plantor portion thereof of claim 51 or method of claim 53, wherein saidportion comprises a plant seed.
 55. The method of any one of claims 1-5,nucleic acid construct of claim 19, plant cell of claim 50 or plant ofclaim 51, wherein said plant is a dicotyledonous plant.
 56. The methodof any one of claims 1-5, nucleic acid construct of claim 19, plant cellof claim 50 or plant of claim 51, wherein said plant is amonocotyledonous plant.
 57. The method of any one of claims 1-5, nucleicacid construct of claim 19, plant cell of claim 50 or plant of claim 51,wherein said plant comprises corn.
 58. The method of any one of claims1-5, nucleic acid construct of claim 19, plant cell of claim 50 or plantof claim 51, wherein said plant comprises sorghum.
 59. The method of anyone of claims 1-5, nucleic acid construct of claim 19, plant cell ofclaim 50 or plant of claim 51, wherein said plant is selected from thegroup consisting of Arabidopsis, sorghum, corn, tobacco, cauliflower,soybean, alfalfa, peach, white spruce, wheat, sugar beet, sunflower,sugarcane, cotton, barley, tomato, potato, oat, carrot and grape.